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1St. Erik's Eye Hospital, Karolinska Institutet, 2Gullstrand lab, Section for Ophthalmology, Department of Neuroscience, Uppsala University
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Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.
Galichanin, K., Talebizadeh, N., Söderberg, P. Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract. J. Vis. Exp. (69), e4016, doi:10.3791/4016 (2012).
Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which impedes the transfer of light. Cataract is a multi-factorial disease associated with diabetes, smoking, ultraviolet radiation (UVR), alcohol, ionizing radiation, steroids and hypertension. There is strong experimental 2-4 and epidemiological evidence 5,6 that UVR causes cataract. We developed an animal model for UVR B induced cataract in both anesthetized 7 and non-anesthetized animals 8.
The only cure for cataract is surgery but this treatment is not accessible to all. It has been estimated that a delay of onset of cataract for 10 years could reduce the need for cataract surgery by 50% 9. To delay the incidence of cataract, it is needed to understand the mechanisms of cataract formation and find effective prevention strategies. Among the mechanisms for cataract development, apoptosis plays a crucial role in initiation of cataract in humans and animals 10. Our focus has recently been apoptosis in the lens as the mechanism for cataract development 8,11,12. It is anticipated that a better understanding of the effect of UVR on the apoptosis pathway will provide possibilities for discovery of new pharmaceuticals to prevent cataract.
In this article, we describe how cataract can be experimentally induced by in vivo exposure to UVR-B. Further RT-PCR and immunohistochemistry are presented as tools to study molecular mechanisms of UVR-B induced cataract.
1. Exposure to Ultraviolet Radiation
3. Quantitative RT-PCR
None of the samples has to reveal any DNA specific PCR products on 1.5 % agarose gel electrophoresis.
4. Immunohistochemical Staining
The various sources of variation in the measurements were estimated with an analysis of variance and it was found that considering three measurements per animal the variance for measurements was on the order of 15 % of that for animals. Thus, considering whole lens analysis, it is not possible to increase the precision. Orthogonal testing elucidated a statistically significant contrast for caspase-3 message between 120 hr latency interval versus shorter latency intervals.
In vivo UVR exposure induces caspase-3 expression.
Figure 1. Schematic of the flow of exposure to ultraviolet radiation.
Figure 2. Evolution of caspase-3 (casp3) mRNA expression in the crystalline lens after in vivo exposure to 1 kJ/m2 UVR at 300 nm. Error bars are 95 % confidence intervals for mean ratio of casp3 mRNA/18s rRNA between exposed lens and contralateral not exposed lens. The means, above and below the black line at 1 rel. unit, respectively, represent up- and down-regulation of the casp3 gene.
Figure 3. Caspase-3 expression (A) in an exposed lens, 24 hr after exposure and (B) in a non-exposed lens. Arrows show labeled cells.
This paper describes methods that enable studying molecular events occurring during UVR-B induced cataract.
Considering, that most information available for in vivo UVR induced cataract was derived from experiments on albino Sprague-Dawley rats 7, 16, 17, 18, 19, we decided to use the albino Sprague-Dawley rat in the current study. Age of the rats was six week old. The gender was chosen to be female because, in contrast to males, females have less allergenic urine. Moreover, there is no difference in gender in relation to severity of UVR induced cataract 20.
All animals were kept and treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Ethical permission was obtained from the Uppsala Animal Experiments Ethics Committee, protocol number C 29/10. Rats are obtained from a commercial breeder (Taconic, Denmark).
A narrowband UVR source was chosen in order for the UVR to be spectrally well defined. Maximum sensitivity of the rat lens to UVR-B is around 300 nm. The dose 1 kJ/m2 was chosen to be below the threshold dose 15. The exposure time of 15 min was selected to induce maximum damage to the lens 21. The dose of exposure to UVR-B and post-exposure time can vary depending on the experimental design.
Paired organ eye has its advantage in terms of statistics and ethics in use of animals in research. Unilateral exposure of the eye enables to use one side as exposed and another side as control in one animal thus it reduces number of animals by half compared to unpaired organ.
In this protocol we used anesthesia as a method for animal immobilization. However, other alternative, rat restrainer 13, which we designed in our laboratory, can be used for immobilization of unanaesthetized animals. Rats have to be conditioned to the rat restrainer prior the UV exposure. This device allows well controlled repeated exposures when anesthesia is not recommended due to its side effects. Here, we used the rat restrainer as a positioning and holding device for anesthetized animals.
The rat restrainer is made of wood and is available in several items in our laboratory. We clean our restraining device before each animal is positioned on it. Wood blocks, guttering and wood shelters are used as enrichment in the rat cages. Such enrichment is approved by Uppsala Animal Experiments Ethics Committee and Federation of European Laboratory Animal Science Associations (FELASA).
The lens has to be dissected in BSS in short period of time (5-10 min). This restriction allows the lens to stay clear and transparent.
The time of 30 min for keeping the lens in the RA1 lysis buffer is enough to disrupt the lens capsule and cortex. The lens nucleus remains hard within 30 min. The lens nucleus, or organelle-free zone is considered to be a transcription non-active part of the lens. Therefore, the nucleus is removed from the sample in order to increase the signal/noise mRNA expression ratio.
The DNA specific primer used for RNA purity (absence of DNA) control was selected arbitrarily to be p53-primer. Any generally occurring coded sequences of DNA of particular animal genome could be chosen. Both lenses were analyzed with PCR for 10 animals per post-exposure interval and for each lens, caspase-3 RNA content was determined in three independent measurements.
RT-PCR enables to measure the mRNA of interest. The reverse transcriptase converts mRNA into complementary DNA which is then amplified by PCR. Measurements were quantified using the standard curve obtained by amplifying serially diluted samples of the cDNA of interest. The advantages of applying a standard curve are that the standard curve provides a reliable way to calculate the uncertainty of the concentration, and that the standard curve provides quantitative measurements of the mRNA of interest. Alternatively, the relative content of the mRNA of interest could be estimated by directly comparing the number of cycles required to obtain a standardized fluorescence signal, the Ct-method. The main drawback of the calibration curve is that it requires usage of more genomic products than the Ct method.
Immunohistochemistry was used to study spatial distribution of active caspase-3 in lens epithelial cells.
The eyes were frozen immediately to -70 °C to stop all ongoing biochemistry. The eyes were then stored at the same temperature for preservation.
Immunohistochemistry is associated with two general problems; the specificity of the primary antibody and non specific binding of the antibody. The antibody should be acquired from a well controlled source, thus guaranteeing the specificity. If possible, tissue containing the epitope should be stained as a positive control. In order to outrule non-specific staining, if possible, the epitope should be blocked before staining with the specific antibody, negative control. Further, non specific staining can be minimized by establishing the optimal antibody concentration and reaction time. By staining both tissue exposed to UVR and tissue not exposed to UVR, it is possible to establish the UVR induced epitope, here caspase-3. To facilitate the counting of cells expressing caspapse-3 signal the fluorescence microscope image was digitally recorded. In order to minimize variation of background noise, we used fixed settings for all microscope photographs.
The intensity of signal to background fluorescence varies on a continuous scale. Therefore, a threshold for significant staining has to be set. However, the average fluorescence may vary spatially within the section observed making it difficult to use an absolute threshold for significant staining. Therefore, usually the judgment of specific staining relies on an experienced observer and the absolute outcome of specific staining depends on the opinion of the experienced observer but will be consistent for that observer. For this reason, only one experienced observer was used. To improve signal caused by the experimental variable, exposure to UVR, as compared to noise, the photograph of each section was counted three times.
If possible, immunohistochemistry should be verified by western blot thus confirming the existence of epitopes with the expected molecular size.
No conflicts of interest declared.
This work was supported by the Karolinska Institutet KID-funding, Swedish Radiation Protection Authority, Karolinska Institutet Eye Research Foundation, Gun och Bertil Stohnes Stiftelse, St. Erik's Eye Hospital Research Foundation, Ögonfonden, Konung Gustav V:s och Drottning Viktorias Frimurarstiftelse.
|Oculentum Simplex||Apoteket, Sweden||336164||5 g|
|Balanced salt solution||Alcon||0007950055|
|3.5 ul β-mercapt–thanol|
|NucleoSpin RNA II total RNA isolation kit||Macherey-Nagel GmbH&Co, Duren, Germany||740955.50|
|p53 DNA specific primers||biomers.net GmbH||Custom made|
|Taq DNA polymerase, dNTPack||Roche||04 728 866 001|
|Ethidium bromide solution 0.5 mg/ml||Sigma||E1385|
|Nano-Drop ND-1000 spectrophotometer||NanoDrop Products|
|1st Strand cDNA synthesis kit for RT-PCR (AMV)||Roche Diagnostics GmbH||11 483 188 001|
|iCycler MyiQ Single Color Real Time PCR detection system||Bio-Rad Laboratories|
|TaqMan Gene Expression Master Mix||Applied Biosystems||4369016|
|TaqMan Gene Expression Assay for caspase 3||Applied Biosystems||Rn00563902_m1|
|TaqMan Gene Expression Assay for 18s||Applied Biosystems||Hs99999901_s1|
|MyiQ software||Bio-Rad Laboratories|
|Cleaved caspase-3 (Asp175)||Cell signaling technology||9661|
|Anti rabbit IgG||Abcam||Ab6798|
|Universal Microscope Axioplan 2 Imaging||Carl Zeiss|
|TBE buffer 10X||Promega||V4251|
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