This video demonstrates a method for inducing cataracts using ultraviolet radiation in the rat. The molecular mechanisms involved in cataract development are subsequently studied using quantitative real-time PCR and immuno cyto immunochemistry. First one, rat's eye is exposed to ultraviolet radiation to induce cataract formation.
The other eye serves as a control. Next, the lenses are dissected and processed to obtain RNA to assess CAS B'S three expression levels by quantitative real-time PCR or stained, using an antibody against the cleaved caspase. Three to assess the expression and localization of active protein.
Ultimately, the results from these assays confirm an increase in caspase three mRNA and expression of the active protein in the nucleus of the lens cells following exposure to UVR. The main advantage of this protocol over existing method is that it use a unique narrow band, UVR source and universal rat restrain allowing us to safely and precisely expose animalized with well-defined UV spectrum and dosage. Moreover, we applied modern biological techniques, quantitative R-T-P-C-R, and immunohistic chemistry To study apoptosis In the lens, Dodi method can provide information about molecular mechanisms of ultraviolet radiation induced cataract.
It can also be applied to other experimental models such as ultraviolet, radiation induced skin cancers, rat behavioral neuroscience, and experimental eye research with focus on apoptosis. Begin this protocol by placing an anesthetized female Sprague dolly rat in a rat restrainer and without squeezing the trunk, tighten the belts until the rat is immobilized. To induce midis or dilation of the pupil, instill one to two drops of a 10 milligram per milliliter solution of tpic aide in each eye.
Then apply ointment on the non-exposed eye and shield it with black tape to block uv. Next, position the animal in the restrainer so that the uncovered eye is positioned to receive a narrow beam of ultraviolet radiation at 300 nanometers with 10 nanometer full width at half maximum generated from a high pressure mercury lamp. Expose the rat unilaterally to a sub-threshold dose one kilojoule per meter squared of UVRB for 15 minutes after exposure.
Remove the tape from the non-exposed eye and apply ointment to both eyes. Return the animal to its cage and cover it with soft paper to keep it warm. Monitor the animal until it has regained sufficient consciousness to maintain sternal recumbent.
Then place the recovered rat back into its cage with others. 1 5 24 and 120 hours. Follow following exposure.
Place the enucleated eye from the euthanized rat cornea down posterior side up on a soft paper tissue. Then working under a dissecting microscope. Use a 27 gauge cannula to punch a minimal hole pushing tangentially to the sclera to avoid damaging the lens that is very close to the sclera.
Next, using a pair of ophthalmic surgery, scissors cut circumferentially just behind the limbus until the posterior portion of the sclera can be lifted off. Then using blunt curved forceps, lift the lens, place the lens into a balanced salt solution and remove remnants of the ciliary body from the lens. The lens should not be kept in the balanced salt solution for more than 10 minutes.
Following dissection, place a lens into a two milliliter micro centrifuge tube containing 350 microliters of ra, one lysis buffer from the nucleo spin RNA two total RNA isolation kit and 3.5 microliters of Beamer keto ethanol Incubate for 30 minutes at room temperature. After incubation, use a needle to mechanically homogenize the lens until only the hard nucleus is left. The cortex and capsule of the lens will be lyed by the lysis buffer.
Remove the nucleus from the mix and discard it. Store samples immediately at negative 70 degrees Celsius. Samples can be stored for up to one year when ready to proceed.
Father samples and purify the RNA according to the total RNA, purification from cultured cells and tissue protocol from the kit. Store the RNA samples at negative 70 degrees Celsius. To verify sufficient removal of DNA from each sample, amplify the DNA using P 53 DNA specific primers under the following conditions.
Step 1 95 degrees Celsius for two minutes. Step two for 40 cycles, 95 degrees Celsius for 20 seconds. 55 degrees Celsius for 20 seconds.
72 degrees Celsius for 20 seconds. Step 3 72 degrees Celsius for seven minutes. Once the run has completed, run the products on a 1.5%AROS gel.
To detect the DNA specific PCR product of 243 base pairs, be sure to include a positive and negative control sample. If the DNA has been successfully removed, the PCR products will not be seen. A band should only be seen in the positive control lane using NanoDrop spectrophotometer to measure the concentration of RNA samples and determine ratio of RNA to protein.
If the ratio RNA to protein of RNA sample absorbance or 2 62 80 ratio is 2.0 or more than the RNA sample is pure. Next, using one microgram of RNA synthesize CD NA using a first strand CD NA synthesis kit for R-T-P-C-R store CD NA samples at negative 20 degrees Celsius. Next to perform quantitative realtime PCR apply the samples in triplicate to a 96 well plate.
Each well should contain one microliter of CD NNA sample combined with TAC man gene expression, master mix tac man gene expression assay for caspase three on a separate plate. Apply the same samples, but using the TAC man gene expression assay for 18 s. Next to generate a standard curve for analysis dilute in series one microliter of CDNA from three randomly chosen non-exposed lenses.
Run serial dilutions together with the CDNA from samples. In a 96 well plate on each plate. Spin down the plate, then run it on an I cycler my iq, single color real-time PCR detection system.
Following the run, using my IQ software. Establish a standard curve expressing number of cycles at threshold as a function of relative concentration of calibrator for the serial dilutions in each plate. Use the standard curve to determine the relative concentration of the sample CDNA measured using the threshold number of cycles for each sample.
Finally, determine the expression level of the target genes by relating the relative concentration of the target cDNA to the internal control 18 s cDNA. To perform immunohistochemical staining, dissect the eye and put it in PBS following dissection. Transfer the eye to a tube filled with 4%para formaldehyde incubate on ice for 20 minutes.
Using a micro pipette, remove the paraform aldehyde. Then transfer the eye to a two milliliter einor tube containing one milliliter of ice cold PBS and incubate for 20 minutes on ice. Next, transfer the eye to an einor tube containing 30%sucrose.
Incubate at four degrees Celsius overnight. The next day, use forceps to transfer the eye to a cup filled with optimal cutting temperature or OCT medium. Put the eye in a straight position for sectioning.
Then place the cup on dry ice to freeze store it minus 70 degrees Celsius until use to section the eye for analysis by fluorescence microscopy. Begin by positioning the OCT embedded eye on a chuck in the cryostat for cryosectioning. Try to align the eye lens flattest side parallel to the edge of the knife.
Cut three five micron thick midsagittal sections from a central portion of each lens. Discard at least six sections between sequential sections to avoid staining the same nuclei in different sections. Once a section has been cut, gently place a slide on top of it.
The section should then stick to the slide. Leave the slides to air dry before use. Slides can be stored at negative 70 degrees Celsius until required for staining.
Allow the slides to return to room temperature. Then antibody stain to identify cleaved CAS phase three as described in the accompanying document within a few hours, inspect the slides under a fluorescence microscope. Count all lens epithelial cell nuclei.
Record the number of all lens epithelial nuclei by counting the DAPI stained cells and the number of caspase three positive nuclei by counting the active caspase. Three labeled cells. Count the cells three times for each section.
Caspase three is a main protease in the execution stage of apoptosis. Transmission electron microscopy. Studies support that UVR induces apoptosis in the lens.
Experimental studies on apoptosis confirm that caspase three is a reliable marker of apoptosis. To determine whether in vivo UVR exposure induces caspase three expression mRNA expression in the crystalline lens was measured by QPCR 1 5 24, and 120 hours following in vivo exposure of one kilojoule per meter square UVR at 300 nanometers. The mean ratio of CASP three, Mr.NA to 18 S-R-R-N-A between exposed lens and the contralateral not exposed lens was then plotted for each time point.
This figure depicts the caspase three mRNA expression over time and demonstrates transcription of apoptosis. Marker caspase three in the lens in vivo after exposure to far UVR orthogonal testing resulted in a statistically significant contrast for caspase three mRNA signal between 120 hours latency interval versus shorter latency intervals. Caspase three expression in an exposed and non-exposed lens was also compared by immunofluorescence microscopy as shown in these images 24 hours after exposure, the nuclei are labeled indicating the presence of active caspase three.
This immunostaining method provides valuable information on spatial distribution of epithelial cells labeled with apoptosis marker caspase three in the lens. After watching this video, you should have a good understanding of how to experimentally induce in V-V-U-V-R cataract and to study molecular mechanism of five UVR induced cataract using quantitative for TPCR and immunohistochemistry.
