For histopathological studies, It is necessary to make whole animal perfusion on the laboratory experimental animals, i.e. rat or mice. What are the parameters can be considered to confirm that, the perfusion is perfect. Presently, I observe the liver for its paleness. I am interested to know about the other clues, which employ macroscopic confirmation about ideal transcardial perfusion of the animals.
Tissues become comparatively harder after ideal transcardial perfusion
The main symptom you have to detect and to keep under surveillance the progressive change in the consistence of the body. The body becomes progressively rigid. The arms and posterior members raised and become extended. These signs indicate that fixative solution has taken the circulation way and penetrates deeply in tissues. At the final step, all the body becomes rigid. If you haven't this state of rigidity, it means that little part of fixative reached the tissues. As postulated above too, the dark color of some organs as liver, kidneys,..turn into pale one.
"For histopathological studies, It is necessary to make whole animal perfusion on the laboratory experimental animals, i.e. rat or mice"
This is not true. You don't need to perfuse an animal to get good tissue preservation for histopathology. As long as the tissues are taken quickly after euthanasia, and an adequate tissue:fixative ratio is used, a simple immersion in formalin is sufficient for most needs.
As veterinary pathologist I can say that for general histopathology whole body perfusion is not a good choice because it can falsify part of the evaluation (eg. there is no congestion) or morphometric measurements. However, for some tests, eg. the neurohistopathology perfusion is necessary in order to minimize changes in the nervous system.
Properly performed perfusion makes animal stiff, the tissues and internal organs are getting pale, and the body (especially after the opening of the abdomen) smell of the liquid fixative. A good solution is flushed blood vessels with heparin in NaCl solution before the administration of the perfusion fluid.
I think that the question of Annadurai was as: ."What parameters can be considered to confirm the perfect perfusion?" So, I think that most of responses have given the parameters to observe in this sense. Concerning, classical histology and histopathology "senso stricto", I agree with the two last responses in that there is no need of perfusion, notably for periphric organs and tissues.
An excellent description, completed with informative schemata, materials list as well as a demo-video on Whole Animal Perfusion of Rodents (Gage et al, 2012) one can find @
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476408/ (Free Download).
Interesting discussion as the interpretation of the question was not the same for all readers. Oneway or another if perfusion is indicated which not alway is, and the choice depends of the protocol selection of tissues to be collected experimentally and tests to be preformed. To reduce number of animal in a specific study the study director may have the option of sharing tissues for various tests. In that case pathologists can do histopathology of internal organs tissue collected ASAP and fixed by immersion. Other may prefer to collect tissues and freeze. Not the best option for histopathology resolution but could be better for other procedures such as histochemistry. Regarding neuropathology assessment yes transcardiac perfusion is ideal. Again study protocol design should indicate, selection of fixative, post-fixation method and fixative, which may vary depending the size and age of the experimental animal selected. Assuming animal model is adult rat, it is best to post fix for 48-72 hours in situ, after opening the skull. Grossing should be done using molds suitable for brain size to best obtain the anatomical levels. Histologically blood vessels should not have blood, be open, maintain the integrity of the BBB and specific cells types such as neurons, astrocytes etc. Perfused brains are better for detail histopathology, as artefacts are kept to the minimum. Enclosed are some examples and publication. Diagnostic neuropathology for veterinary and human medical procedures perfusion is not an option, but at the same time tissues changes are usually more visible. Whereas, experimental neuropathology, and even more developmental studies, tissues changes may be minimal and need to be compared agains controls. may consist in changes of cell migration, myeline distribution, evaluation of neuroneogenesis etc.
Hi,
In our lab we frequently do the perfusion, Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed for fixation. Tissue become harder as well as you will see the tail of mice or rat will be stiff. you will also found the heart and kidney pale and i think 20ml 4% PFA is enough for the perfect perfusion.
Thank you.
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