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, &Ouml;gonfonden, Konung Gustav V:s och Drottning Viktorias Frimurarstiftelse.

1. Exposure to Ultraviolet Radiation
<ol>
	<li>15 min before the exposure, anesthetize a female Sprague-Dawley rat with a mixture of 90 mg/kg Ketalar (ketamine) and 10 mg/kg Rompun (xylazine) by intraperitoneal injection.</li>
	<li>Place the animal in a rat restrainer and tighten the belts until immobilization of the rat without causing a trunk squeeze 13.</li>
	<li>Instill Mydriacyl (tropicamide), 10 mg/ml, in both eyes of the rat to induce mydriasis.</li>
	<li>Place the animal so that one eye is positioned...

<ol>
	<li>Brian, G., Taylor, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cataract+blindness:+challenge+for+the+21+st+century.">Cataract blindness: challenge for the 21 st century.</a> Bulletin of the World Health Organization. 79, 249-256 (2001).</li><li>Jose, J. G., Pitts, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelength+dependency+of+cataracts+in+albino+mice+following+chronic+exposure.">Wavelength dependency of cataracts in albino mice following chronic exposure.</a> Experimental Eye Research. 41, 545-563 (1985).</li><li>S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+cataract+in+the+rat+after+exposure+to+radiation+in+the+300+nm+wavelength+region.+A+study+of+the+macro-,+micro-+and+ultrastructure.">Acute cataract in the rat after exposure to radiation in the 300 nm wavelength region. A study of the macro-, micro- and ultrastructure.</a> Acta Ophthalmol. (Copenh). 66, 141-152 (1988).</li><li>Meyer, L., Dong, X., Wegener, A., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose+dependent+cataractogenesis+and+Maximum+Tolerable+Dose+(MTD+2.3:16)+for+UVR+-+B+induced+cataract+in+C57BL/6J+mice.">Dose dependent cataractogenesis and Maximum Tolerable Dose (MTD 2.3:16) for UVR - B induced cataract in C57BL/6J mice.</a> Experimental Eye Research. 86, 282-289 (2008).</li><li>McCarty, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lifetime+ocular+exposure+to+UV-B:+the+Melbourne+visual+impairment+project.">Assessment of lifetime ocular exposure to UV-B: the Melbourne visual impairment project.</a> Developments in Ophthalmology. 27, 9-13 (1997).</li><li>Sasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+cortical+cataract+in+subjects+of+diverse+races+and+latitude.">Localization of cortical cataract in subjects of diverse races and latitude.</a> Invest. Ophthalmol. Vis. Sci. 44, 4210-4214 (2003).</li><li>S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+cataract+induced+by+ultraviolet+radiation.">Experimental cataract induced by ultraviolet radiation.</a> Acta Ophthalmol. (Copenh. 68, 1-77 (1990).</li><li>Galichanin, K., L&#246;fgren, S., Bergmanson, J., S&#246;derberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+damage+in+the+lens+after+in+vivo+close+to+threshold+exposure+to+UV-B+radiation:+cytomorphological+study+of+apoptosis.">Evolution of damage in the lens after in vivo close to threshold exposure to UV-B radiation: cytomorphological study of apoptosis.</a> Exp. Eye Res. 91, 369-377 (2010).</li><li>McCarty, C. A., Taylor, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+epidemiologic+evidence+linking+ultraviolet+radiation+and+cataracts.">A review of the epidemiologic evidence linking ultraviolet radiation and cataracts.</a> Dev. Ophthalmol. 35, 21-31 (2002).</li><li>Li, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens+epithelial+cell+apoptosis+appears+to+be+a+common+cellular+basis+for+non-congenital+cataract+development+in+humans+and+animals.">Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals.</a> Journal of Cell Biology. 130, 169-181 (1995).</li><li>Michael, R., Vrensen, G., van Marle, J., Gan, L., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+the+rat+lens+after+in+vivo+threshold+dose+ultraviolet+irradiation.">Apoptosis in the rat lens after in vivo threshold dose ultraviolet irradiation.</a> Investigative Ophthalmology and Visual Science. 13, 2681-2687 (1998).</li><li>Ayala, M. N., Strid, H., Jacobsson, U., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+Expression+and+Apoptosis+In+The+Lens+After+Ultraviolet+Radiation+Exposure.">p53 Expression and Apoptosis In The Lens After Ultraviolet Radiation Exposure.</a> Investigative ophthalmology of visual science. 48, 4187-4191 (2007).</li><li>Galichanin, K., Wang, J., Lofgren, S., Soderberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+universal+rat+restrainer+for+ophthalmic+research.">A new universal rat restrainer for ophthalmic research.</a> Acta Ophthalmol. 89 (1), (2011).</li><li>Ayala, M. N., Michael, R., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cataract+after+repeated+exposure+to+ultraviolet+radiation.">In vivo cataract after repeated exposure to ultraviolet radiation.</a> Experimental Eye Research. 70, 451-456 (2000).</li><li>S&#246;derberg, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+ultraviolet+radiation+exposure+to+the+lens+expressed+by+maximum+tolerable+dose+(MTD).">Toxicity of ultraviolet radiation exposure to the lens expressed by maximum tolerable dose (MTD).</a> Developments in Ophthalmology. 35, 70-75 (2002).</li><li>Michael, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+repair+of+cataract+induced+by+ultraviolet+radiation.">Development and repair of cataract induced by ultraviolet radiation.</a> Ophthalmic Research. 32, 1-45 (2000).</li><li>L&#246;fgren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cataract from ultraviolet radiation. , (2001).</li><li>Ayala, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Influence of exposure patterns and oxidation in UVR induced Cataract. , (2005).</li><li>Dong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Safety limit estimation for cataract induced by ultraviolet radiation. , (2005).</li><li>L&#246;fgren, S., Michael, R., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+age+and+sex+in+ultraviolet+radiation+cataract+in+the+rat.">Impact of age and sex in ultraviolet radiation cataract in the rat.</a> Investigative Ophthalmology and Visual Science. 44, 1629-1633 (2003).</li><li>Ayala, M. N., Michael, R., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+exposure+time+for+UV+radiation-induced+cataract.">Influence of exposure time for UV radiation-induced cataract.</a> Investigative Ophthalmology and Visual Science. 41, 3539-3543 (2000).</li></ol>

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...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Ketalar</td>
			<td>Pfizer</td>
			<td>150086</td>
			<td>50 mg/ml</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td>022545</td>
			<td>20 mg/ml</td>
		</tr>
		<tr>
			<td>Oculentum Simplex</td>
			<td>Apoteket, Sweden</td>
			<td>336164</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Mydriacyl</td>
			<td>Alcon</td>
			<td>00352</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>Balanced salt solution</td>
			<td>Alcon</td>
			<td>0007950055</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 ul &beta;-mercaptoethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin RNA II total RNA isolation kit</td>
			<td>Macherey-Nagel GmbH&amp;Co, Duren, Germany</td>
			<td>740955.50</td>
			<td></td>
		</tr>
		<tr>
			<td>p53 DNA specific primers</td>
			<td>biomers.net GmbH</td>
			<td>Custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA polymerase, dNTPack</td>
			<td>Roche</td>
			<td>04 728 866 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A5093</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution 0.5 mg/ml</td>
			<td>Sigma</td>
			<td>E1385</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano-Drop ND-1000 spectrophotometer</td>
			<td>NanoDrop Products</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1st Strand cDNA synthesis kit for RT-PCR (AMV)</td>
			<td>Roche Diagnostics GmbH</td>
			<td>11 483 188 001</td>
			<td></td>
		</tr>
		<tr>
			<td>iCycler MyiQ Single Color Real Time PCR detection system</td>
			<td>Bio-Rad Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Sarstedt</td>
			<td>72.1979.202</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4369016</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay for caspase 3</td>
			<td>Applied Biosystems</td>
			<td>Rn00563902_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay for 18s</td>
			<td>Applied Biosystems</td>
			<td>Hs99999901_s1</td>
			<td></td>
		</tr>
		<tr>
			<td>MyiQ software</td>
			<td>Bio-Rad Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaved caspase-3 (Asp175)</td>
			<td>Cell signaling technology</td>
			<td>9661</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti rabbit IgG</td>
			<td>Abcam</td>
			<td>Ab6798</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>84097</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Microscope Axioplan 2 Imaging</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>441244</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE buffer 10x</td>
			<td>Promega</td>
			<td>V4251</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of molecular mechanisms of in vivo uvr induced cataract

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.

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 ...

Watch this Scientific Journal Video about Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract at JoVE.com

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

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.

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

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 ...

characterization-molecular-mechanisms-vivo-uvr-induced

Research

JoVE Journal

Medicine

9.7K visualizações.  Karolinska Institutet. A catarata é a principal causa de cegueira no mundo. Radiação solar ultravioleta (UVR) é o principal fator de risco para o desenvolvimento de catarata. Um modelo animal de longe RUV-B catarata induzida foi desenvolvido. Neste artigo vamos descrever os métodos de investigação de formação de catarata: a exposição a raios ultravioletas, RT-PCR e imuno-histoquímica.