This protocol allows the study of spinal cord regeneration in frogs. The knowledge acquired could provide novel insights to understand how humans respond to spinal cord injury. The main advantage of this protocol is the easiness in performing the injury, and also that compared to the paradigm of tail amputation, this technique mimics better what happens in humans.
This technique allows to challenge the regenerative process to better understand the participation of different genes, proteins, molecules, and signaling pathways, thereby identifying new therapeutic targets, The finding the right place for generating the injury, as well as its extension, is difficult. Being able to see those steps is crucial for making this procedure straightforward and reproducible. Three to four weeks after fertilization, place the tadpoles in a Petri dish and check the morphology and appearance of the fore limbs and hind limbs.
Look for the following anatomical characteristics of stage-50 animals, fore limbs that are just appearing and are spherical, and hind limbs that are protruding and are spherical. Before the surgery, anesthetize the tadpoles by placing them in 0.02%tricaine mesylate in 0.1 times Barth solution, and check that the animals are not responsive by turning them upside down. To perform the spinal cord transection surgery, use a tablespoon and forceps to place an anesthetized stage-50 tadpole dorsal side up on a wet piece of gauze in the upper-half of a glass Petri dish.
Then, using microdissection spring scissors, make an incision of the skin and dorsal muscles at the midthoracic level. For control sham animals, ensure that the incision size is only 0.2 millimeters and does not damage the spinal cord. In the transected animals, make a second incision of 0.2 millimeters to fully transect the spinal cord.
After surgery, transfer the tadpoles to a tank containing 500 milliliters of 0.1 times Barth solution with one times penicillin and streptomycin at a density of 10 to 12 tadpoles per tank. Maintain the transected and control sham tadpoles in separate tanks. Maintain the tadpoles with aeration at a temperature of 20 to 21 degrees Celsius.
Change the Barth solution with antibiotics every other day until the end of the experiment. Start feeding the tadpoles one day after surgery and eliminate the dead animals. For the swimming assay, obtain a box with LED illumination from the inside, covered with a transparent polystyrene sheet allowing light to pass through, and install a camera over the LED box.
Then, place a 15-centimeter-diameter Petri dish on top of the box filled with 100 milliliters of 0.1 times Barth solution. One day post-transection, place a tadpole in the Petri dish and leave it for a five-minute adaptation period. After adaptation, start video tracking the free-swimming behavior using the reference software for five minutes.
After the video is completed, transfer the tadpole back to its tank. This representative dot plot shows the recovery of motor function through time. Five days post-transection, tadpoles swam an average of 0.7 meters in five minutes, showing a reduced swimming capacity.
This capacity increased with passing days, showing an average of 2.1 meters in five minutes after 10 days, and 3.1 meters in five minutes after 15 days post-transection. Complete recovery of swimming capacities was observed at 20 days post-transection, with an average of 5.7 meters in five minutes. The swimming average can vary depending on each batch of animals.
Thus, the experimental group should always be compared to the respective controls and not previously obtained data. After surgery, animals can be used for global analysis, including transcriptomics, proteomics, and metabolomics, and to screen for compounds that inhibit or promote spinal cord regeneration, aiming to develop new therapeutic approaches.
