Cryoinjury Induction in Fish Hearts

Fish were anesthetized with 0.02% Tricaine (MS-222) and transferred to a moist sponge for surgery. After visually locating the posterior medial margin of the heart straight iridectomy scissors were used to puncture the skin and the silvery pericardial sac. Subsequently, an incision was made through both the skin and pericardium starting from the junction of the pericardium and peritoneum and reaching anteriorly for about 2/3 of the length of the heart. The incision was spread open laterally using fine forceps to expose the ventricle. Small pieces of dry ice were formed into a conical shape with a length of ∼20 mm, one end with a diameter of ∼2 mm and the other end with a pointed tip. The pointed tip of the dry ice cone was applied to the posterior apex of the ventricle for 10 seconds to cause the cryoinjury. After surgery the fish were returned to holding tanks. To revitalize the fish a pipette was used to vigorously squirt water over the gills until the fish started to breathe regularly. Sham treated control fish in which the pericardial sac was opened but the heart left untouched showed no signs of necrosis, induction of wt1b:GFP expression or upregulation of cardiomyocyte proliferation. Thus, hearts of untreated fish served as uninjured control samples in most experiments.