Transcardiac perfusion is a specialized technique used in scientific research to prepare biological samples. It involves flushing the circulatory system of an animal with various solutions to clear blood and preserve tissues. This method is employed to obtain high-quality organs and tissues that are well-preserved for subsequent detailed study and analysis.
What is Transcardiac Perfusion?
Transcardiac perfusion clears blood from an animal’s circulatory system, replacing it with preserving solutions. This technique is performed after humane euthanasia or deep anesthesia. Its goal is to obtain clean, well-preserved organs and tissues for analysis, free of blood components.
The underlying principle involves introducing a perfusate, a specialized solution, into the vascular system. This perfusate circulates throughout the blood vessels, washing out blood cells and debris. The process helps maintain cellular integrity and prevent tissue degradation. An initial washout with saline or phosphate-buffered saline (PBS) prepares the tissues for a fixative, ensuring optimal preservation.
Why is Transcardiac Perfusion Performed?
Transcardiac perfusion achieves superior tissue preservation and clarity. By removing blood, researchers avoid artifacts and interference that blood cells might cause in microscopic examinations and biochemical analyses. This leads to more accurate and reliable data.
In neuroscience, transcardiac perfusion is used for brain studies. Blood removal ensures clear images for immunostaining or in situ hybridization, allowing visualization of neuronal structures and connections. This technique also benefits toxicology studies, where precise tissue concentrations of drugs or compounds need to be determined. For histology, perfusion yields high-quality tissue sections free of cellular debris and blood, allowing detailed microscopic examination and ensuring structural integrity.
How is Transcardiac Perfusion Performed?
The transcardiac perfusion procedure begins with anesthetizing the animal to a surgical plane. The animal is positioned on its back, and an incision exposes the chest cavity. A small incision is then made in the right atrium for perfusate and blood outflow.
A needle or cannula is inserted into the left ventricle of the heart. Through this cannula, a washout solution, typically a physiological saline solution, is pumped through the circulatory system. This initial flush removes blood. As blood is flushed, organs, such as the liver, visibly turn pale.
Following the saline flush, a fixative solution, commonly 4% paraformaldehyde (PFA), is introduced through the same cannula. The fixative circulates throughout the body, preserving tissues by cross-linking proteins and DNA, which maintains cellular and tissue structure. The procedure is complete when the animal’s body becomes stiff and organs like the liver show a pale brownish color, indicating thorough fixation.
Ethical Aspects and Animal Welfare
Transcardiac perfusion is conducted under strict ethical guidelines to ensure animal welfare. The procedure is always performed on animals that are deeply anesthetized or humanely euthanized prior to perfusion.
Institutional Animal Care and Use Committees (IACUCs) provide oversight for all animal research, including transcardiac perfusion. These committees review and approve protocols to ensure animal use is scientifically justified and adheres to humane standards. Personnel involved must have training and competency in performing the procedure and administering anesthesia.