We are going to show you how to prepare Japanese quail ex ovo culture and how to use chorioallantoic membrane or CAM in short for photodynamic diagnosis and therapy of cancer or microbial infections. Advantages of this method are rapid developmental growth, small size, good access to the tissue, and easy handling. Also, it respects the principles of the 3R rule for conducting animal research.
Due to its structural similarity to the mucous membranes such as the bladder, lungs, or placenta, it is suitable for in vivo testing of potential drugs, toxicity testing, or transplantation studies. Demonstrating the procedure will be Barbora Kundekova and Majlinda Meta, PhD students from my laboratory. To begin, use fertilized quail eggs, fresh or stored at 10 to 15 degrees Celsius for a maximum of 4 to 5 days prior to the start of incubation.
Use only clean and undamaged eggs, then incubate the eggs in a forced-draught incubator at 50 to 60%humidity and 37.5 degrees Celsius for approximately 54 hours placed horizontally with the egg rotations switched off. Disinfect the egg surface with 70%ethanol without rotating the egg. Next, in a sterile laminar flow cabinet while wearing gloves, open the eggshell using small sterile surgical scissors and transfer the content into a 6-well culture plate.
After each egg, disinfect the scissors with 70%ethanol. Add approximately 5 milliliters of sterile water to the gaps in the 6-well plate as humidity is essential to prevent the CAM from drying out. Next, aspirate improperly-tipped embryos or unfertilized eggs with a vacuum aspirator, then place the embryos in an incubator until further experiments while maintaining a temperature of 37 degrees Celsius in 80 to 90%humidity.
When the CAM is developed enough, usually from embryonic day 7, place a sterilized silicone ring on the CAM surface along the small capillaries while avoiding major blood vessels. Under sterile conditions, apply an appropriate volume of hypericin solution 30 microliters to the silicone ring and keep the embryos in an incubator at 37 degrees Celsius in 80 to 90%humidity. To conduct photodynamic diagnosis, illuminate the CAM using violet excitation light and record the fluorescence of hypericin and CAM tissue and tumor cells with a digital camera at different time intervals after hypericin administration.
If the research requires an image of the CAM in white light, record the CAM before hypericin administration and at the end of the experiment shortly before the tissue fixation, as the light triggers photo activation of hypericin. To perform the photodynamic therapy after hypericin application, place the CAM under the optical fiber for the laser beam to cover the entire area within the silicone ring. After performing in vivo irradiation, in this particular case with a 405 nanometers laser light at a fluence rate of 285 milliwatts per centimeter square, record CAM using white light and/or fluorescent light before and after photodynamic treatment.
Fix the CAM tissue in a cultivation plate with 4%paraformaldehyde in PBS for a minimum of 2 hours and a maximum of overnight, then remove the paraformaldehyde and carefully cut out from the CAM a part of the tissue within the silicone ring. All these steps should be done in a fume hood. Wash cut part from the CAM tissue in water for 10 minutes and subsequently dehydrate in ascending alcohol series by placing the CAM tissue in 70%ethanol for 3 minutes, eosin solution for 2 minutes, throcin 96%ethanol for 5 minutes, 100%ethanol for 5 minutes, and twice in xylene for 10 minutes in a new Petri dish.
Subsequently, transfer the samples as quickly as possible to the dissolved paraffin in Petri dishes using a spatula or thin brush. After 24 hours, place the tissue in histological mold, fill it with a paraffin embedding medium and allow it to solidify in the refrigerator, then cut the solidified CAM from the embedding medium, turn it in the tray by 90 degrees, fill it again with the embedding medium and let it solidify. Prepare 5 to 10 micrometer sections on a microtome for histopathological analysis to determine PDT-induced damage.
To prepare the frozen CAM sections for histology, carefully mount native or 4%paraformaldehyde fixed CAM on the glass slide, fill the embedding mold to half with OCT and freeze in liquid nitrogen or a mixture of dry ice and ethanol. After freezing, carefully tilt and slide the CAM from the glass slide onto the top of the frozen OCT medium. Place again in the mold, cover it with OCT medium and freeze as described previously.
For the analysis of CAM vasculature, whole CAM samples are needed. In the laminar flow cabinet, overflow CAM with a prewarmed fixation solution of 4%paraform aldehyde, and 2%glutaraldehyde in PBS, then remove the fixation solution after 48 hours. Next, carefully separate the CAM from the embryo with micro scissors and a fine brush and wash it in PBS, then mount the washed CAM on a glass slide and let it slowly dry.
Afterward, photograph the slide using a digital camera in a transilluminator as a source of homogenous white light. The location of the tumor on the CAM surface was visualized under white light and fluorescent light upon the addition of hypericin. Histological analysis showed concentric structures of abnormal squamous cells invading the healthy tissues accompanied by edema and membrane thickening.
After treatment with PDT, a clear difference in vessel density inside the ring and the surrounding area was observed. The laser radiation without the presence of photosensitizer did not cause any damage comparable to the control without any treatment. After 3 hours of incubation with hypericin, laser irradiation resulted in fluorescence caused by monomerization of the aggregated hypericin with extensive damage to the vasculature.
Histological analysis revealed that untreated control CAM had relatively even thickness. However, PDT treatment caused the CAM to become thinner and more fragile. We just demonstrated how to prepare quail ex ovo CAM assay and how to use it.
While maintaining the guidelines, this methodology is easy to master and can prove quick results.
