Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Konstantin Galichanin; Nooshin Talebizadeh; Per Söderberg

doi:10.3791/4016

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Podsumowanie
Streszczenie
Protokół
Wyniki
Dyskusje
Ujawnienia
Podziękowania
Materiały
Odniesienia
Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

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.

Protokół

1. Exposure to Ultraviolet Radiation

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.
Place the animal in a rat restrainer and tighten the belts until immobilization of the rat without causing a trunk squeeze ¹³.
Instill Mydriacyl (tropicamide), 10 mg/ml, in both eyes of the rat to induce mydriasis.
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.
Expose the rat unilaterally to sub-threshold dose 1 kJ/m² UVR-B at 300 nm for 15 min ¹⁵.

2. Dissection

After pre-determined post exposure interval (1, 5, 24 and 120 hr), sacrifice the six week old rat (weight < 200 g) by carbon dioxide asphyxiation and dislocation of the cervical vertebrae.
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.
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.

3. Quantitative RT-PCR

Place a lens into a mix of 350 μl RA1 lysis buffer (NucleoSpin RNA II total RNA isolation kit) and 3.5 μl β-mercaptoethanol in a 2 ml Eppendorf tube.
Keep the lens in this mix during 30 min at room temperature.
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.
Store samples immediately at -70 °C.
Thaw the samples and follow the protocol "Total RNA purification from cultured cells and tissue" (NucleoSpin RNA II total RNA isolation kit).
Store RNA samples at -70 °C.
To verify sufficient removal of DNA from each sample, run a PCR under the following conditions (step 1: 95 °C for 2 min; step 2 for 40 cycles: 95 °C for 20 sec, 55 °C for 20 sec, 72 °C for 20 sec; step 3: 72 °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.
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 μl in 500 ml of 1.5 % agarose gel solution. Heat the mixture in a microwave owen and stir to solve the agarose.

None of the samples has to reveal any DNA specific PCR products on 1.5 % agarose gel electrophoresis.

Measure the concentration of RNA samples (1 μl) 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.
Take a volume of RNA sample corresponding to 1 μg and synthesize cDNA following the protocol 1^st Strand cDNA synthesis Kit (1^st Strand cDNA synthesis Kit for RT-PCR).
Store cDNA samples at -20 °C (for a period of 1 and more years, store at -70 °C).
Run quantitative real-time PCR on iCycler MyiQ Single Color Real Time PCR detection system. Load triplicates on 96-well plate with 1 μl 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 μl 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.
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.

4. Immunohistochemical Staining

Fixation
1. Dissect the eye in PBS (Phosphate Buffered Saline; pH 7.4).
2. 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 °C until use. PFA is fresh for approximately one week.
3. Remove PFA with a micropipette. Then, wash with ice cold PBS for 20 min.
4. Put the eye in sucrose 30 % at +4 °C over night.
5. Put the eye in a cup filled with Optimal Cutting Temperature (OCT)-medium (Tissue-Tek). Put the eye in a suitable position for sectioning.
6. Freeze in OCT-medium over dry ice.
7. Store at -70 °C until use.
Sectioning
1. Position the eye for cryo-sectioning in an appropriate plane.
2. Cut three 5 μm 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.
3. Once a section has been cut, gently place a slide on top of it. The section should then stick to the slide.
4. Leave the slides to air-dry before use. Slides can be stored at -20 °C until required.
Fluorescence immunohistochemistry
1. Let samples attain room temperature (5-10 min).
2. Draw edges around the specimens with a PAP-pen (Invitrogen).
3. Let the border dry for a while (10 min).
4. Label the microscope slides.
5. Rehydrate in 1 × PBS for 15 min.
6. Permeabilize in standard block solution for 30 min.
7. Add primary antibody (rabbit polyclonal, cleaved caspase-3 antibody Asp175 9661; Cell Signalling Technology, Inc) diluted 1:3,000 in standard block solution.
8. Store in a humidified chamber at +4 °C over night.
9. Wash in PBS by pipetting (3 × 5 min) or by immersing in a large bath (>15 min, 1-2 changes).
10. Add secondary antibody (anti rabbit secondary antibody with a specific absorption/emission spectrum) diluted 1:300 in the standard block solution.
11. Wash in PBS by pipetting (3 × 5 min) or by immersing in a large bath (>15 min, 1-2 changes).
12. Wipe off PAP-pen smears.
13. Store in a humidified chamber at room temperature for 3 hr. Avoid exposure to light.
14. Add a drop of Vectashield and place a cover slip on the slide. Avoid making bubbles.
15. Let the Vectashield harden (~20 min).
16. Keep sections in the dark until analysis.
17. All control sections are processed in the absence of primary antibody.
18. Look for the results within a few hours under a fluorescence microscope.
19. 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.
20. Record the number of all lens epithelial nuclei and the number of positive nuclei. Count the cells three times for each section.