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Improvements in the technique of vascular perfusion‐fixation employing a fluorocarbon‐containing perfusate and a peristaltic pump controlled by pressure feedback
Author(s) -
ROSTGAARD J.,
QVORTRUP K.,
POULSEN S. S.
Publication year - 1993
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1993.tb03405.x
Subject(s) - glutaraldehyde , fixative , peristaltic pump , perfusion , fixation (population genetics) , chemistry , cannula , biomedical engineering , chromatography , surgery , biochemistry , medicine , physics , cytoplasm , meteorology , cardiology , gene
Summary A new, improved technique for whole‐body perfusion‐fixation of rats and other small animals is described. The driving force is a peristaltic pump which is feedback regulated by a pressure transducer that monitors the blood/perfusion pressure in the left ventricle of the heart. The primary perfusate‐fixative is composed of a blood substitute—13·3% oxygenated fluorocarbon FC‐75—in 0·05 M cacodylate buffer (pH 7·4) with 2% glutaraldehyde. The secondary perfusate‐fixative is composed of 2% glutaraldehyde in 0·05 M cacodylate buffer (pH 7·4) with 20 mM CaCl 2 . A double‐barrelled, self‐holding cannula is used to cannulate the heart; the outer and inner barrels of the cannula are connected to the peristaltic pump and to the pressure transducer, respectively. The tissue oxygen tension in the rat is monitored by a subcutaneous oxygen electrode. Measurements showed that tissue hypoxia/anoxia did not develop before or during the perfusion‐fixation. Thus, the technique permits study of specimens which do not exhibit fixation gradients and do not contain cells fixed in a state of asphyxia. This is substantiated by electron micrographs of cells from different organs, revealing new fine structural elements. By adding oxygenated fluorocarbon to glutaraldehyde perfusate‐fixatives, enough oxygen is made accessible for cellular respiration as well as for the oxygen‐consuming chemical reactions of glutaraldehyde with the tissue. Data on anaesthesia, operative manoeuvres, mechanical components of the system, preparation of fixatives and flow of the perfusate‐fixatives are furnished and discussed.