Surgical Ablation Assay for Studying Eye Regeneration in Planarians

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

The overall goal of this procedure is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues so that the technique can be implemented by researchers and students at all levels of education. This method can help answer key questions in the regenerative field, such as determining the mechanisms that regulate endogenous eye stem cell maintenance and differentiation in vivo. The main advantage of this technique is that it allows to researchers to remove just the eye tissues with minimal disturbance of other tissues.

Generally, practice is required before individuals new to this method can reliably remove the correct amount of tissue, ablating the entire eye while avoiding ablation of surrounding tissues. To begin, select worms that have not been fed for at least one week and are at least five to seven millimeters long. Transfer 15 to 30 worms to a 100 millimeter Petri dish two-thirds full of worm water and immediately replace the cover.

Observe the worms for missing or unpigmented tissues, such as white heads or tails, to ensure that they are undamaged and not recently regenerating. First, wipe the workspace and dissecting microscope base with a 70%ethanol solution, and let it dry completely. Place the dish with the selected worms on the left side of the microscope.

Then, on the right side of the microscope, place a pair of number five forceps, a 31 gauge 5/16ths inch insulin needle with a one milliliter syringe, a clean transfer pipette, and a labeled 12-well plate for collecting ablated worms. Place a box of lint-free tissue wipes, a plastic wash bottle filled with fresh worm water, and additional transfer pipettes so that they are easily accessible during the surgery. Position a Peltier plate at the center of the dissecting microscope base, and set the output on the DC power source so that the surface of the Peltier plate is sufficiently cool to immobilize worms.

Alternatively, an ice-filled Petri dish can be used in place of a Peltier plate. To prepare the surgery surface, first cut a five centimeter by ten centimeter piece of plastic paraffin film and place it over the center of the Peltier plate. Next, fold a lint-free tissue wipe into a square of approximately two centimeters, and place it on top of the film.

Then, hold the folded wipe in place, moisten it with worm water, and roll the wipe flat with a transfer pipette. Finally, cut a piece of white filter paper of 1.5 to two centimeters squared, and lay it on top of the wipe. To begin the surgery, place one worm dorsal side up onto the filter paper, using the transfer pipette.

Turn the light on and focus its beam on the worm. Adjust the dissecting microscope focus and magnification so that the eyes of the worm are clearly visible. Rotate the paraffin film to adjust the position of the worm so that its head is pointed towards the researcher and angled 30 to 40 degrees to the right.

If the worm is positioned ventral side facing up, and the pharyngeal opening is visible, use the dull side of the forceps to gently change the position of the worm to dorsal side up with the eyes visible. Readjust the microscope settings so that the eyes are clearly in focus. Also ensure that both the eyes and surrounding head tissues are in view.

Hold a clean syringe in the right hand between thumb and index finger. Support the bottom part of the syringe on the left thumb, braced against the Peltier plate. Look through the microscope to ensure the bevel of the needle is visible.

Place the left index finger on the surgery surface so that it makes a 40-degree angle with the left thumb to ensure that the surface remains stable during the procedure. Position the needle at a 90-degree angle to the eye with the bevel of the needle facing upward. With the tip of the needle, gently penetrate the thin layer of tissue overlying the optic cup of the eye visible as white, unpigmented region.

Scooping from right to left, very gently remove any black pigmented tissue located within the optic cup as well as all of the white tissues. If performing double eye ablation, ablate the second eye now. After completing the surgery, backload the pipette with a small amount of worm water, and release the water onto the worm to lift it off the filter paper.

Immediately draw the worm into the transfer pipette. Move the worm to a labeled 12-well plate two-thirds full of fresh worm water. Once all worms are transferred, wash them by replacing the water from the wells with fresh worm water.

Incubate the worms protected from light in a 20-degree Celsius incubator and monitor the process of regeneration. To sacrifice live worms, remove worm water from the plate and replace it with 70%ethanol. Incubate the worms for three to five minutes and observe whether the worms lyse and turn gray.

Presented here are photographs of flatworms subjected to double eye ablation and single eye ablation, taken before, four days after, and two weeks after the surgical procedure. The images demonstrate regeneration progress over time. Eye regeneration in ablated worms was also confirmed by immunohistochemical staining of arrestin and newly developed photoreceptor neurons.

The recovery of the visual system in flatworms following double eye ablation was also studied in a functional assay based on the wildtype photophobic response to light. 24 hours after the ablation, eyeless worms do not avoid light and travel through light spots. Photophobia is restored by seven days after the procedure, indicating that the regenerated visual system is functional.

Once mastered, the procedure of a double eye ablation of one worm can be done approximately three minutes. When ablating, it's important to excise all of the pigmented and unpigmented tissues of the eye, while making sure not to damage the tissue between the eyes or to tear the tissue later into the eye. Following this procedure, other methods like immunohistochemistry and RNA interference can be performed in order to examine the role of specific genes and their regenerative process.

Additionally, behavioral assays can be performed to test eye function following regrowth. After watching this video, you should have a good understanding of how to excise planarian eyes without disturbing surrounding tissues. Don't forget that working with needles can be hazardous, and precautions such as wearing gloves and holding the needle pointing away from you should be taken.