The overall goal of this procedure is to demonstrate our perfusion fixation technique that emphasizes the use of physiological pressures to properly preserve rat brain tissue for immunohistochemistry procedures. First, the use and setup of the perfusion apparatus is demonstrated. Then the tools needed to perform both the perfusion surgery and the brain removal are assembled, and the deeply anesthetized animal is placed in the shallow tray filled with crushed ice.
A five to six centimeter lateral incision through the integument and abdominal wall is made just beneath the rib cage. The heart is exposed and the animal is perfused. The final step is to remove the brain and place it in a vial of fixative containing fluid at least 10 times the volume of the brain itself.
The overall goal of this procedure is to demonstrate our profusion fixation technique that emphasizes the use of physiological pressures to properly preserve rat brain tissue for immunohistochemistry, We will start by preparing the perfusion rig. This will take about 10 minutes. Using a 50 milliliter syringe repeatedly flush and clear the fixative line with buffer until all air bubbles are removed.
It is crucial for the success of a perfusion not to have air bubbles in any of the lines. This is achieved by placing the outlet valve into a beaker filled with buffer. Now, turn the exit valve off.
Then remove the tube from the syringe while squeezing it so a drop of buffer hangs from the end. Place the filled tubing, the fixative inlet into a bottle with 200 milliliters of paraldehyde fixative per animal. Taking care not to introduce any air bubbles.
Tighten the cap, making sure the seal is properly seated. If it is not seated properly, the pressure within the closed system will not hold. Place the bottle into the perfusion rig.
Set the flow from only the buffer line by closing the outlet port and turning the buffer valve to the same position as the fixative valve. Open the outlet port and clear the buffer line as done before for the fixative line. Once the line is cleared, place the tube into the buffer bottle.
Next, test the system's ability to hold pressure. By pumping the rubber manometer bulb, there will be resistance. As a final preparation.
Fill the perfusion needle with buffer to prevent the introduction of air. The system is now ready for perfusion. Having administered a ketamine xylazine mixture unchecked for a surgical plane of anesthesia via the toe pinch method, place the fully anesthetized rat on a shallow tray filled with crushed ice.
Do not continue unless the animal is fully anesthetized. Begin the surgery with a five to six centimeter lateral incision through the integument and abdominal wall just beneath the rib cage. Carefully separate the liver from the diaphragm.
Make a small incision in the diaphragm using the curved blunt scissors. The position and pressure of your finger can aid in the ability to cut the diaphragm. Continue the diaphragm incision along the entire length of the rib cage to expose the pleural cavity.
Place curved blunt scissors along one side of the ribs, carefully displacing the lungs and make a cut through the rib cage up to the collarbone. Then make a similar cut on the contralateral side. Now lift the sternum away and carefully trim any tissue, connecting it to the heart.
Clamp the tip of the sternum with the hemostat and place the hemostat over the head. The Thur should lift away providing a clear view of the major vessels. Using iris scissors, make a small incision to the posterior end of the left ventricle.
Pass a 15 gauge blunt or olive tipped perfusion needle through the cut ventricle into the ascending aorta. The tip should be visible through the wall of the aorta and should not reach the aortic arch where the brachial and carotid arteries diverge. To secure the needle and prevent leakage, clamp the heart with a hemostat.
Finally, use the iris scissors to make a large outlet in the right atrium. Be careful to not damage the descending aorta. At this point, the animal is ready to be perfused.
Attach the perfusion needle in the right atrium to the outlook port without introducing any air bubbles and open the outlook port. Then quickly and evenly pump up the manometer bulb to a pressure of 80 millimeters of mercury. Maintain this pressure throughout the buffer infusion period.
Now start a timer. Adjust the needle angle to where the flow is strongest, and when the buffer is almost finished, switch the buffer valve. The fluid will begin running clear when the liver is perfused.
This is an indication of a good perfusion and the time of the liver clearing should be noted very quickly After this event, fixation tremors will be observed. Indicate this as the time of true fixation. To maintain a steady flow rate, the pressure can be gradually increased to a maximum of 130 millimeters of mercury.
Once the fixative is nearly finished, close the outlet valve and take a note of the time. The rat should now be stiff. The rat's organs may now be harvested To remove the brain, begin the dissection of the fixed rat by removing the head using a guillotine.
Next, make a midline incision along the integument from the neck to the nose and expose the skull with scissors, orals. Trim off the remaining neck muscle so that the base of the skull is exposed. Place the sharp end of a pair of iris scissors into the for and magnum on one side, carefully sliding the scissors along the inner surface of the skull.
Next, make a cut extending to the distal edge of the posterior skull surface. Make an identical cut on the contralateral side. Then use theros to clear away the skull around the cerebellum.
Carefully slide the tip of the scissors along the inner surface of the skull from the dorsal distal posterior corner to the distal frontal ledge. Avoid damaging the brain by lifting up on the tip of the blade. During this process, repeat this, cut on the opposite side.
Then using raws, peel the dorsal surface of the skull away from the brain. Trim away the sides of the skull using raws as well. Now using a spatula, sever the olfactory bulbs and nervous connections along the ventral surface of the brain and gently tease the brain away from the head.
Using iris scissors, cut any dura connecting the brain to the skull. Once the brain is removed, place it in a vial of fixative that is at least 10 times the volume of the brain itself. Store the vial at four degrees Celsius for 24 hours.
After 24 hours, wash the brain with PB S3 times with gentle swirling. The brain can then be stored in PBS or heis Hanks. With sodium azide at four degrees Celsius, brains can be stored for three months.
Un sectioned. Once mastered, the entire perfusion could be completed between five and eight minutes.
