A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Summary

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Abstract

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers Müller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Introduction

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers Müller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Protocol

All procedures described in this protocol were approved by the animal use committee at Wayne State University School of Medicine.

1. Dark Adaptation

1. Transfer ~10 adult albino or pigmented fish from the normal housing system into a dark enclosure. If available, use a dark enclosure that is built into the zebrafish housing module, which allows for normal water flow through the tank. (If such a system is not available, place the fish tank in a completely dark enclosure, making sure to aerate the fish with oxygen).
2. Keep the fish in the dark for 10 days. When feeding the animals or adding new fish to the dark box, make sure to move as quickly as possible to avoid exposing the fish to a long period of light.

2. UV Light Exposure

1. Make sure the power to the UV source is OFF. Remove the UV light filament from fluorescent stereomicroscope.
   1. Use an inverted 15 cm diameter glass Petri dish (or something of a similar 2 cm height) as a stand for the UV filament. Tape the Petri dish to the lab bench.
   2. Tape the UV light filament to top of the inverted Petri dish. Arrange so that ~2 mm of the end of the filament overhangs the Petri dish.
2. Obtain a 250 ml glass beaker. Cover ½ of the bottom, sides, and back of the beaker with aluminum foil, making sure the "shiny" side of the foil faces the interior of the beaker. If the beaker is graduated, cover the worded half of the beaker with foil, leaving the clear half of the beaker exposed.
3. Fill the 250 ml beaker with 100 ml of water from the fish facility system.
4. Place the 250 ml beaker in a 4 L beaker. Fill the 4 L beaker with water until the water level is even with the 100 ml water line in the 250 ml beaker.
5. Add a maximum of 10 dark-treated animals to the 250 ml beaker. Cover the 250 ml beaker with a small piece of aluminum foil.
6. Place the entire beaker apparatus immediately adjacent to the UV filament. The filament should be touching the outside of the 4 L beaker and facing the exposed portion of the 250 ml beaker. Make sure that the 250 ml beaker is centered in the bottom of the 4 L beaker.
7. Position a large, opaque screen behind the 4L beaker, allowing for the animals to be exposed, but preventing any lab personnel from seeing the tip of the UV filament. WARNING: make sure this barrier is in place BEFORE turning on the power to the UV source.
8. Turn on the UV power source. Set timer for 30 min. Place whatever necessary warning labels to ensure that unsuspecting lab personnel do not accidentally expose themselves to UV radiation.
9. After 30 min, shut off the UV power source. Remove the 250 ml beaker with the fish. NOTE: if multiple rounds of UV exposure are required, let the power source cool down (~20 min) before repeating the exposure protocol. Make sure to replace the 100 ml of water with fresh system water for each exposure. The water in the 4 L beaker does not need to be replaced.

3. Halogen Lamp Light Exposure

1. Transfer the fish from 250 ml beaker into a 1.8 L clear acrylic fish tank. Fill the tank to the overflow mark.
2. Set up the halogen lamp light treatment area. Obtain four 250 W halogen utility lamps from a local hardware store. Facing two lamps in the same direction, arrange 29 cm apart on center. Arrange the other two lamps in a similar fashion.
   1. Place the second set of lamps so that they are facing the first set of lamps, leaving ~73 cm in between the two sets of lamps. This creates a rectangle-shaped light treatment area of 29 x 73 cm.
3. Obtain a small fan from a local hardware store. Place the fan equidistant between the two sets of lamps, just outside of the light treatment area.
4. Place two 1.8 L tanks full of water in the center of the light treatment area, equidistant between the two sets of lamps. The distance between the lamps and the outside wall of the tank should be ~29 cm. Note: even if only treating 10 fish in one 1.8 L tank, use this arrangement. Both tanks are needed to keep the water temperature of each tank within the appropriate range.
5. Place an oxygen aerator in each tank.
6. Cover each tank with clear acrylic lids, leaving a ~2 cm gap at the end closest to the fan. Arrange the fan so that it will blow air into this gap. Place a thermometer in one or both of the tanks.
7. Turn on the power to the lights, fan, and aerators. Maintain light treatment for up to 4 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.
8. Monitor temperature and water level on a daily basis. Maintain the temperature at 30-33 °C. If necessary, adjust the fan speed and/or the distance between the tanks and the lights.
   1. Top off each tank with system water as needed, usually daily.
   2. Always use healthy adult zebrafish (typically 6-9 months of age) to maintain a survival rate of near 100%. However, if a fish is found dead in the tank, remove it immediately and replace the water in the entire tank.

4. Tissue Collection

1. 48 hr after the onset of the halogen light treatment remove the fish from the treatment area.
   1. Prepare fresh ethanolic formaldehyde fixative (1 part 37% formaldehyde, 9 parts 100% ethanol).
   2. Euthanize zebrafish with an anesthetic overdose of 2-phenoxyethanol (0.4 mg/L).
   3. Transfer the euthanized zebrafish to a paper towel. Enucleate the eye using curved forceps.
   4. Place enucleated eyes in fixative and store O/N at 4 °C.
2. Cryoprotect the eyes.
   1. Wash the eyes in 5% sucrose 1x PBS for 30 min at room temperature, then replace with fresh 5% sucrose 1x PBS for 2 hr. Next, wash the eyes in 30% sucrose 1x PBS O/N at 4 °C. Wash the eyes in a 1:1 (equal portions) of tissue freezing medium and 30% sucrose 1x PBS O/N at 4 °C.
3. Embed the eyes in 100% tissue freezing medium and store at -80 °C. Orient the eyes so that cryosectioning of the tissue is performed on the dorsal/ventral axis.
4. Cryosection the tissue and place on glass slides. Warm slides for 2 hr at 55 °C. Store slides at -80 °C or immediately perform standard immunohistochemistry.
5. Perform standard immunohistochemistry on sectioned tissue and image with fluorescent microscopy^40,51.

Results

The heretofore described light treatment protocol was compared to each individual method of LIRD. In dark-treated adult albino animals (Figures 3-5), the individual light treatments resulted in significant loss of rod (Figure 3) and cone (Figure 4) photoreceptors. However, both individual treatments primarily damaged photoreceptors in the dorsal half of the retina, leaving the ventral retina relatively protected from the light treatments (...

Discussion

Here we show that combining a short UV exposure with a continual bright light exposure results in widespread photoreceptor loss and a robust regeneration response. Compared with the individual LIRD methods, this combined method is also the most effective protocol to damage both rods and cones in both halves of the retina. Importantly, this treatment is effective in pigmented animals as well as albino animals.

Although we provide evidence that the combined protocol results in more wide...

Materials

Name	Company	Catalog Number	Comments
UV light source	Leica	EL600
Glass Petri dish (150 x 20 mm)	Sigma-Aldrich/Pyrex	CLS3160152BO
250 ml glass beaker	Sigma-Aldrich/Pyrex	CLS1000250
4 L glass beaker	Sigma-Aldrich/Pyrex	CLS10004L
Aluminum foil	Fisher	01-213-105
250 W halogen lamps	Workforce	265-669
1.8 L clear acrylic tanks	Aquaneering	ZT180T
1.8 L clear acrylic tank lids	Aquaneering	ZT180LCL
Fan	Honeywell	HT-900
Aerator	Tetra	77853-900
Thermometer	Cole-Parmer	YO-08008-58
Bent forceps (5/45)	World Precision Instruments	504155