The overall goal of this procedure is to combine two previously described light induced retinal degeneration protocols in order to damage both rod and cone photoreceptors throughout the retina. This is accomplished by first exposing dark adapted adult zebra fish to UV light for 30 minutes. The second step is to immediately transfer the UV treated fish into tanks surrounded by four, 250 watt halogen lamps and expose the fish to continuous light for up to four days.
The final step is to euthanize the fish and nucleate the eyes for further analysis. Ultimately, immunofluorescence microscopy is used to show consistent loss of both rod and cone photoreceptors throughout the retina. The main advantage of this technique over existing methods like a UV light treatment or halogen lamp like treatment, is that combining these two methods results in the ablation of both rod and cone photoreceptors throughout the retina.
Damage to the photoreceptors induces a widespread proliferation response that ultimately results in regeneration of the loss photoreceptors. Demonstrating this technique will be Jennifer Thomas, a graduate student in my laboratory. To begin, use an inverted 15 centimeter diameter glass Petri dish for use as a stand and tape it to the lab bench for UV light exposure.
Remove the UV light filament from the fluorescent stereo microscope. Then tape the UV light filament above the inverted Petri dish and allow about two millimeters of the end of the filament to overhang the Petri dish. After that, cover half of the bottom sides and back of a 250 milliliter glass beaker with aluminum foil, and to make sure that the shiny side of the foil faces the interior of the beaker.
Next, fill the beaker with 100 milliliters of water from the fish facility system. Place this beaker in a four liter beaker. Then fill the four liter beaker with water until the water level is even with the 100 milliliter waterline in the 250 milliliter beaker.
At a maximum of 10 dark treated fish to the 250 milliliter beaker, covered the 250 milliliter beaker with a small piece of aluminum foil. Next place the entire beaker apparatus immediately adjacent to the UV filament. The filament should be touching the outside of the four liter beaker and facing the exposed portion of the 250 milliliter beaker.
Make sure that the 250 milliliter beaker is centered at the bottom of the four liter beaker. Afterward, position a large opaque screen behind the four liter beaker, which allows the animals to be exposed, but prevents any lab personnel from seeing the tip of the UV filament. Then turn on the UV power source.
Set the timer for 30 minutes and place necessary warning labels to ensure that unsuspecting lab personnel do not accidentally expose themselves to UV radiation. After 30 minutes, shut off the UV power source. Then remove the 250 milliliter beaker with the fish.
In this procedure, transfer the fish from the 250 milliliter beaker into a 1.8 liter clear acrylic fish tank. Next, fill the tank to the overflow mark to set up the halogen lamp light treatment area. Place two, 250 halogen utility lamps in the same direction and 29 centimeters apart from the tank.
Then place the other two lamps in a similar fashion on the opposite side, leaving about 73 centimeters in between the two sets of lamps. After that, place the fan equidistant between the two sets of lamps just outside of the light treatment area. Subsequently, place two 1.8 liter tanks filled with water in the center of the light treatment area, equidistant between the two sets of lamps.
Then place an oxygen aerator in each tank. Cover each tank with clear acrylic lids, leaving a two centimeter gap at the end closest to the fan. Arrange the fan so that it will blow air into this gap, and place a thermometer in one or both of the tanks.
Next, turn on the power of the lights fan and aerators. Maintain the light treatment for up to four days. The fish should not be fed during the light treatment, as this will affect water quality and result in more stress to the animals.
Monitor the temperature and water level on a daily basis and maintain the temperature at 30 to 33 degrees Celsius. If necessary, adjust the fan speed the distance between the tanks and the lights. Also fill each tank with system water daily.
48 hours after the onset of the halogen light treatment, remove the fish from the treatment area. Next, prepare fresh ethanol formal to hide fixative euthanize the zebra fish with an anesthetic overdose of tuen oxy ethanol. After that, transfer the euthanized zebra fish to a paper towel, then nucleate the eye using a pair of curved forceps.
Keep the nucleated eyes and fixative overnight at four degrees Celsius to cryo. Protect the eyes. Wash them in 5%sucrose one XPBS for 30 minutes at room temperature.
Then replace it with fresh 5%sucrose one XPBS and keep the eyes for two hours. After two hours, wash the eyes in 30%sucrose, one XPBS overnight at four degrees Celsius. The next day, wash the eyes in a one-to-one tissue, freezing medium and 30%sucrose, one XPBS solution overnight at four degrees Celsius.
Afterward, embed the eyes in 100%tissue freezing medium oriented on the dorsal ventral axis. Then store them at minus 80 degrees Celsius for at least two hours before cryosectioning the tissue. Now cryo section the tissue and place it on glass slides.
Then warm the slides for two hours at 55 degrees Celsius. After that, store the slides at minus 80 degrees Celsius or immediately perform standard immunohistochemistry and image with fluorescent microscopy. This figure shows the changes in proliferation response following various light damage paradigms.
Here are the retinal sections from an adult albino zebrafish, immuno labeled with PCNA to show cell proliferation indicated in red in the dorsal and ventral halves of the retina following different light damage paradigms at zero hours. Proliferation was absent from the interclear layer at 48 HLT. Single progenitor cells were present in the INL across the dorsal retina, and in a small portion of the ventral retina.
At 48 HPUV. Columns of progenitor cells were present in the dorsal retina while only single progenitor cells were present in a small portion of the ventral retina. At UV plus 48, large columns of progenitor cells were present across the dorsal and ventral retina.
This figure shows rod and cone photoreceptor loss in pigmented zebra fish following various light damage paradigms. Here are the retinal sections stained with to Pro three to show the nuclei in blue and immuno labeled with zpr R three to show rod outer segments in magenta or zpr R one to show double cones in gold following each of the light damage paradigms. Significant decreases in rod photoreceptor nuclei were present in the dorsal retina at 48 HLT and in the dorsal and ventral retina at UV plus 48 HLT.
Although nuclei were hypertrophied in the dorsal retina at 48 HPUV, no significant decreases in double cone nuclei were present at 48 HLT or 48 HPUV. Significant decreases in nuclei were present in the dorsal and ventral retina at UB plus eight H lt. Combining this procedure with other methods like gene specific pharmacological inhibitors or in vivo protein knockdown can reveal the role of individual genes and photoreceptor degeneration and the subsequent regeneration response.
