In contrast to humans, zebrafish can regenerate their heart after injury. The overall goal of the following experiment is to characterize regeneration of the zebrafish heart in response to myocardial infarction. This is accomplished by inducing heart infarcts using a technique called cryin injury, which destroys approximately 20%of the ventricle after a specific recovery period.
The hearts are collected and fixed, and the progression of cardiac regeneration at different time points is assessed. Then the fixed hearts are sectioned in replicates in order to perform different analyses of the same specimen results are obtained that show various cellular and molecular aspects of the regenerative process using histological, staining, situ to hybridization and immunofluorescence. The main advantage of the reinjury technique of our existing methods in zebrafish, like partial amputation of the ventricular apex is that it causes extensive cell death within a large portion of the myocardium.
In fact, rapid free storing tissue mimics physiological responses of ischemia induced infarction observed in humans. This method can help answer key questions in the field of regenerative biology, such as what factors stimulate cardiomyocyte proliferation in zebrafish. How can the injured heart maintain its life essential contractile activity during extensive tissue remodeling that involves transient scar deposition and regrowth of the lost heart muscle?
How are these opposite cellular responses of fibrosis and new myocardial formation coordinated to achieve complete heart regeneration in zebrafish? The implications of this technique extend toward better understanding why mammals display poor ative capacity when compared to zebra fish. The cryin injury technique will be presented by Fabian Shale, a PhD student who established this technique in our laboratory.Heart.
Sectioning will be then demonstrated by Zimmerman, our technical assistant To prepare for cryosurgery. Have ready a stereo microscope, sharp forceps, micros, dissecting spring scissors, and a plastic transfer pipette. In addition, prepare a beaker for anesthetizing the fish and a plastic spoon for transfer.
The main tool used to perform the surgery is the cryo probe, a pen like instrument made of stainless steel to avoid frostbit while performing the procedure, use a plastic tube and tape to handle the probe for maintaining the fish in a stable position. During the procedure, cut a groove into a moist sponge. Set up a double timer first with a ten second countdown, and then automatically a 24 second countdown.
To begin the cryo injury procedure. First, cool down the cryo probe by immersing the tip in three to five centimeters of liquid nitrogen for at least three minutes. Anesthetize and adult zebra fish by submerging it in 0.02%trica until the fish turns on its back and its gills.
Almost stop moving. Using the plastic spoon, transfer the fish to the groove of the moist sponge with its ventral side up. With the non-dominant hand hold the fish steady with forceps.
Next, visually locate the posterior medial margin of the heart and use straight iridectomy scissors to puncture the skin. Then make a small incision above the heart by cutting straight in an anterior direction through the skin and muscles. With the tip of the scissors, gently tear the silvery epithelial layer of the hypodermis to have direct access to the beating ventricle.
Do not insert the scissors deeper into the body cavity as this will puncture the heart. The beating heart should be well visible and no extensive bleeding should occur during the thoracotomy. Start the programmed timer During the first 10 seconds, remove the cryo probe from the liquid nitrogen and shake it gently to remove any nitrogen.
Then use forceps to open the chest by laterally spreading the incision. Once the ten second timer goes off, immediately and gently use the tip of the pre-cool cryo probe to touch the ventricle. The probe should be precisely positioned on the heart to ensure a tight contact with the ventricle.
To achieve this, the ventricle should be exactly positioned in the center of the incision. When the timer rings after 24 seconds, use the plastic pipette to pour two to three milliliters of system water onto the chest to release the cryo probe from the tissue and transfer the fish into the tank with system water. If it doesn't breathe, after around 45 seconds, stimulate the animal by using a plastic pipette to squirt water into the gills until it starts breathing by itself To collect and fix the heart.
First, prepare one milliliter of 2%formin in a micro fuge tube. Also have ready two forceps, micros, dissecting scissors, and the moist slotted sponge on the selected day. After cryo injury, euthanize the fish in 0.1%trica for five minutes.
Place the fish ventral side up in the sponge using the micros dissecting scissors. Make a large incision above the heart through the brachial cartilage. Use forceps to widely open the incision.
Next, pinch off the bulbus arteriosis a white structure anterior to the ventricle and remove the heart from the cavity by pulling it for fixation. Place up to three hearts into the tube of 2%Formin. Gently turn the tube several times and keep it overnight at four degrees Celsius.
To prepare the hearts for mounting, rinse them in PBS for five minutes. Then transfer them into 10 milliliters of pre cooled, 30%sucrose. Incubate the hearts for one hour and 20 minutes.
At four degrees Celsius, the hearts will sink to the bottom of the tube. Next, prepare a box with dry ice. Then pour a five millimeter layer of OCT mounting medium into the bottom of an embedding mold.
Using forceps, place a heart into the OCT medium in the mold under a stereo microscope. Orient the heart so that the ventricular apex is on the bottom of the mold and the bulbous arteriosis faces up. Then place the mold on dry ice for a few minutes until the OCT freezes.
Then add more OCT to fill up the mold. Set up a cryostat with a cutting size of 16 microns, a chamber temperature of negative 24 degrees Celsius and the temperature of the specimen at negative 22 degrees Celsius. Place a frozen block with a specimen in the cryostat and fix its orientation so that cutting begins parallel to the bottom of the block.
Prepare six super frosts plus slides per block and number them from one to six. Begin cutting until the tissue is reached and then use a razor blade to trim the block around the specimen To obtain six replicates of one heart, take up the first section on slide one, the second section on slide two, the third on slide three, et cetera. Once the sixth section is placed on slide six, restart with slide one and continue until the whole organ is cut.
Collect two rows of eight sections per slide. Let the slides dry for one hour at room temperature. Store them for up to one year in tightly closed boxes at negative 20 degrees Celsius Show shown here is a representative cardiac injury in a dissected whole heart four days post cryo injury.
To visualize the myocardial tissue vivo transgenic fish expressing EGFP and nuclear dazs red two under the control of a cardiac specific CM C two promoter were used. The absence of eeg, FP and DS red, two fluorescent signals demarcated a disc shaped infarct zone along the apical lateral ventricular wall to determine the extent of heart regeneration versus scarring. Histological analysis using acid fusion orange GS staining, which differentially labels cardiac and fibrotic tissues was performed.
Healthy muscle cells are labeled in orange. The scar tissue containing collagen in blue and fibrin in red gene expression analyses were carried out using insight to hybridization. Ventricular myosin.
Heavy chain mRNA from intact myocardium is seen here in blue. The injured tissue is devoid of the cardiac gene transcript. In this figure, an immunofluorescent staining of TRO mycin shown in green labels intact cardiomyocytes and not damaged tissue.
The nuclei are stained with DPI and are shown in blue in this panel, genetically labeled cardiomyocytes express. DS RED two in their nuclei, DAPI labels all the nuclei. However, the infarct zone does not contain DS red.
Two positive cells. Once master read, the cryin injury of a single fish can be done in less than five minutes if it's performed properly. While attempting this procedure, it's important to remember to place the called cryo probe precisely on the vle for the optimal duration After development of the cryo injury technique.
This method paved the way for researchers in the field of regenerative biology to explore mechanisms underlying natural cardiac restoration. After watching this movie, you should have a good understanding how to induce myocardial infarction in CPR fish using cryo injury.
