Improvements in the technique of vascular perfusion-fixation employing a fluorocarbon-containing perfusate and a peristaltic pump controlled by pressure feedback.

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 a 2% glutaraldehyde. The secondary perfusate-fixative is composed of 2% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) with 20 mM CaCl2. 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.