Morphology of the isolated rat hindquarter preparation: tissue preservation, perfusion heterogeneity and a note on the effect of fixative osmolality.

Abstract

The isolated rat hindquarter preparation often used for physiological studies was subjected to morphological examination. A two-hour perfusion with balanced oxygenated dextran solution was followed by perfusion fixation with aldehydes. Fixation was performed at isogravimetry when an isotonic fixative was used (2.5% glutaraldehyde in 0.05 M Na cacodylate + 4% dextran 70). Muscle specimens were prepared for light and electron microscopy. Most muscle tissue was well perfused but a limited tissue volume could retain blood after the perfusion period. Within perfused tissue the perfusion result varied macroscopically with some regions apparently not being reached by fixative. Non-fixed tissue was estimated to amount to maximally one fourth of the total tissue mass. Also within macroscopically well-fixed tissue the microscopy indicated varying patency of perfused capillaries. Thus a marked perfusion flow heterogeneity prevailed in the fixed specimens which is compatible with earlier functional findings of a 50% reduction of capillary surface area during isogravimetric fixation. The possible relation with prefixation flow heterogeneity in the vasodilated preparation is discussed. There were no obvious alterations of endothelial ultrastructure after prolonged artificial perfusion. Isogravimetrically fixed muscle had narrow extracellular spaces which were evidently compressed further after use of absorbing hyperosmolar fixative. Extracellular expansion and intracellular oedema followed after hypo-osmolar fixation. Muscle cells often exhibited insufficient mitochondrial fixation, probably as a result of tissue hypoxia.