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 against a narrow beam of UVR at 300 nm 14 with 10 nm full width at half maximum and shield the contralateral eye with black tape.</li>
	<li>Expose the rat unilaterally to sub-threshold dose 1 kJ/m2 UVR-B at 300 nm for 15 min 15.</li>
</ol>
2. Dissection
<ol>
	<li>After pre-determined post exposure interval (1, 5, 24 and 120 hr), sacrifice the six week old rat (weight &lt; 200 g) by carbon dioxide asphyxiation and dislocation of the cervical vertebrae.</li>
	<li>Enucleate eyes. Thereafter, put the eye cornea down, posterior side up. Then, punch a minimal hole by applying a 27 gauge cannula and push tangentially to the sclera to avoid damaging the lens that is very close to the sclera. Then use a pair of ophthalmic surgery scissors to cut circumferentially just behind the limbus until the posterior portion of the sclera can be lifted off. Then, lift the lens with a blunt curved forceps.</li>
	<li>Remove remnants of the ciliary body from the lens equator under a microscope, keeping the lens in balanced salt solution no longer than 5-10 min.</li>
</ol>
3. Quantitative RT-PCR
<ol>
	<li>Place a lens into a mix of 350 &mu;l RA1 lysis buffer (NucleoSpin RNA II total RNA isolation kit) and 3.5 &mu;l &beta;-mercaptoethanol in a 2 ml Eppendorf tube.</li>
	<li>Keep the lens in this mix during 30 min at room temperature.</li>
	<li>Homogenize the lens with a needle until only hard nucleus of the lens is left from the lens (cortex and capsule of the lens are lysed in the lysis buffer). Remove the nucleus from the mix.</li>
	<li>Store samples immediately at -70 &deg;C.</li>
	<li>Thaw the samples and follow the protocol "Total RNA purification from cultured cells and tissue" (NucleoSpin RNA II total RNA isolation kit).</li>
	<li>Store RNA samples at -70 &deg;C.</li>
	<li>To verify sufficient removal of DNA from each sample, run a PCR under the following conditions (step 1: 95 &deg;C for 2 min; step 2 for 40 cycles: 95 &deg;C for 20 sec, 55 &deg;C for 20 sec, 72 &deg;C for 20 sec; step 3: 72 &deg;C for 7 min) with p53 DNA specific primers, forward 5'-ACCCTCTGACCTTTTTCCCA-3' and reverse 5'-TGCTGGGATCTTAGGCACTC-3' and Taq DNA polymerase (dNTPack), according to the manufacture protocol. The expected PCR product is 243 base pairs.</li>
	<li>Run a 1.5 % agarose gel electrophoresis to detect DNA specific PCR product of 243 base pairs. To prepare 1.5 % agarose gel in TBE buffer take 3.75 g of agarose and solve it in 250 ml of TBE buffer. Add ethidium bromide (0.5 mg/ml) 500 &mu;l in 500 ml of 1.5 % agarose gel solution. Heat the mixture in a microwave owen and stir to solve the agarose.</li>
</ol>
None of the samples has to reveal any DNA specific PCR products on 1.5 % agarose gel electrophoresis.
<ol start="9">
	<li>Measure the concentration of RNA samples (1 &mu;l) and the ratio of sample absorbance at 260 nm (RNA) and 280 nm (protein) in a NanoDrop ND-1000 spectrophotometer. If the ratio RNA to protein of RNA sample absorbance is 2.0 or more then the RNA sample is pure. If the ratio of RNA sample absorbance is lower than 2.0, redo RNA purification step 3.5.</li>
	<li>Take a volume of RNA sample corresponding to 1 &mu;g and synthesize cDNA following the protocol 1st Strand cDNA synthesis Kit (1st Strand cDNA synthesis Kit for RT-PCR).</li>
	<li>Store cDNA samples at -20 &deg;C (for a period of 1 and more years, store at -70 &deg;C).</li>
	<li>Run quantitative real-time PCR on iCycler MyiQ Single Color Real Time PCR detection system. Load triplicates on 96-well plate with 1 &mu;l cDNA sample, TaqMan Gene Expression Master Mix, TaqMan Gene Expression Assay for caspase 3, according to manufacture instructions. Load triplicates on another 96-well plate with 1 &mu;l cDNA sample, TaqMan Gene Expression Master Mix, TaqMan Gene Expression Assay for 18s, according to manufacture instructions (TaqMan Gene Expression Assay Protocol). Dilute in series a fraction of cDNA from three randomly chosen non-exposed lenses. Run serial dilutions together with the cDNA from samples in a 96-well plate.</li>
	<li>Use the standard curve method for quantification of the results. The number of cycles at threshold fluorescence is used as the measurement in MyiQ software. A standard curve expressing number of cycles at threshold as function of relative concentration of calibrator is established for the serial dilutions in each plate. cDNA from three non treated lens samples was used for calibration. Relate the threshold number of cycles for each sample to the standard curve to obtain the relative concentration of the sample cDNA measured. Finally, get expression level of the target genes by relating the relative concentration of the target cDNA to the internal control 18s cDNA.</li>
</ol>
4. Immunohistochemical Staining
<ol>
	<li>Fixation
	<ol>
		<li>Dissect the eye in PBS (Phosphate Buffered Saline; pH 7.4).</li>
		<li>Put the eye in a tube filled with 4 % paraformaldehyde (PFA) in ice cold for 20 min. Prepare PFA 4 % the day before. Store at +4 &deg;C until use. PFA is fresh for approximately one week.</li>
		<li>Remove PFA with a micropipette. Then, wash with ice cold PBS for 20 min.</li>
		<li>Put the eye in sucrose 30 % at +4 &deg;C over night.</li>
		<li>Put the eye in a cup filled with Optimal Cutting Temperature (OCT)-medium (Tissue-Tek). Put the eye in a suitable position for sectioning.</li>
		<li>Freeze in OCT-medium over dry ice.</li>
		<li>Store at -70 &deg;C until use.</li>
	</ol>
	</li>
	<li>Sectioning
	<ol>
		<li>Position the eye for cryo-sectioning in an appropriate plane.</li>
		<li>Cut three 5 &mu;m thick mid-sagittal sections from a central portion of each lens. Discard at least 6 sections between sequential sections to avoid staining the same nuclei in different sections.</li>
		<li>Once a section has been cut, gently place a slide on top of it. The section should then stick to the slide.</li>
		<li>Leave the slides to air-dry before use. Slides can be stored at -20 &deg;C until required.</li>
	</ol>
	</li>
	<li>Fluorescence immunohistochemistry
	<ol>
		<li>Let samples attain room temperature (5-10 min).</li>
		<li>Draw edges around the specimens with a PAP-pen (Invitrogen).</li>
		<li>Let the border dry for a while (10 min).</li>
		<li>Label the microscope slides.</li>
		<li>Rehydrate in 1 &times; PBS for 15 min.</li>
		<li>Permeabilize in standard block solution for 30 min.</li>
		<li>Add primary antibody (rabbit polyclonal, cleaved caspase-3 antibody Asp175 9661; Cell Signalling Technology, Inc) diluted 1:3,000 in standard block solution.</li>
		<li>Store in a humidified chamber at +4 &deg;C over night.</li>
		<li>Wash in PBS by pipetting (3 &times; 5 min) or by immersing in a large bath (&gt;15 min, 1-2 changes).</li>
		<li>Add secondary antibody (anti rabbit secondary antibody with a specific absorption/emission spectrum) diluted 1:300 in the standard block solution.</li>
		<li>Wash in PBS by pipetting (3 &times; 5 min) or by immersing in a large bath (&gt;15 min, 1-2 changes).</li>
		<li>Wipe off PAP-pen smears.</li>
		<li>Store in a humidified chamber at room temperature for 3 hr. Avoid exposure to light.</li>
		<li>Add a drop of Vectashield and place a cover slip on the slide. Avoid making bubbles.</li>
		<li>Let the Vectashield harden (~20 min).</li>
		<li>Keep sections in the dark until analysis.</li>
		<li>All control sections are processed in the absence of primary antibody.</li>
		<li>Look for the results within a few hours under a fluorescence microscope.</li>
		<li>Count epithelial cell' nuclei from one side nuclear bow to other side nuclear bow of each lens. Apply the standard blue filter to see all lens epithelial nuclei in blue and a standard filter that fits the emission of the secondary antibody to see the caspase-3 positive nuclei in green.</li>
		<li>Record the number of all lens epithelial nuclei and the number of positive nuclei. Count the cells three times for each section.</li>
	</ol>
	</li>
</ol>

<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. 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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>

No conflicts of interest declared.

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 ...

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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 ...

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9.8K Views.  Karolinska Institutet. 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.

Video: Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...