Decalcification by perfusion.
new methodfor rapid softening of temporal bones
Magnus Nilsson, Sten Hellstrom and Nils Albiin
Department ofAnatomy and Otolaryngology, Head and Neck Surgery, University of UmeA, UmeA, Sweden
We describe a new technique, decalcificationby perfusion, for the softening of bony tissue. The bloodcirculatory system was perfused in 16 rats via a cannulathrough the left heart ventricle with a fixative followedby New DecalcR (an acidic demineralizer) for 30-240minutes. Perfusion decalcification for 120 minutessoftened al1 heads and middle ear specimens could beeasily sampled and prepared for studies by both light andelectron microscope. For comparison, a conventionalimmersion technique required 72 hours of decalcificationto accomplish softening. The perfusion techniqueconsiderably reduced the time needed to decalcifythe tissue and preserved the morphology better thandid the immersion procedure.
Decalcification, Perfusion, Immersion,Ear, Temporal bone, Rat
The middle and inner ear are enclosed in andprotected by the temporal bone. Dissection ofinteresting portions in order to study their microscopicalarchitecture in light and electron microscopes oftenrequires softening of the tissue. Hitherto, this procedurehas included immersion of thespecimens in variousdifferent decalcifying agents such as both strongand weak organic or mineral acids, acidic buffersand chelating agents, e.g. EDTA (ethylenediamine-tetraacetic acid), (Preece, 1965; Baird et al., 1967;Pearsxe, 1968; Kivaranta et al., 1980).Adequate decalcification by immersion of temporalbones, e.g. from the rat, is time-consuming and requiresabout 3 days in acidic demineralizers and 10-14 days inchelating agents (Anniko, 1981). Furthermore,immersion for long periods in acidic demineralizers
Magnus Nilsson, Department
UmeA, Swedencauses distortion of tissue structures and a reducedaffinity for histological stains (Preece, 1965; Kiviranta etal., 1980).A pilot study (Albiin, 1986) indicated thatadministration of a decalcifier by perfusion, via thecirculatory system of rats, considerably shortened thetime needed for softening. The purpose of the presentstudy was to develop this method and ascertain theoptimal time needed for softening the temporal bones inthe rat. The quaility of the tissue morphology, includingits ultrastructural appearance, and the affinity forhistological stains were evaluated and compared withspecimens decalcified by immersion.
Materials and methods
Twenty-two healthy Sprague-Dawley rats, weighing350-400 g, were used for the study. The animals wereanesthetized with an intraperitoneal injection of sodiumpentobarbital (MebumalR)
mgkg body weight.Through a ventral rnidline incision the thoracic cagewas opened and a cannula inserted into the ascendingaorta via the left heart ventricle. The animal wasthen perfused with a fixative solution containing
glutaraldehyde, 4% polyvinylpyrrolidone (PVP, m01 wt40,000) in 75 mM cacodylate buffer with
alciumchloride added (McDonal and Lame, 1983). The 525mOsm solution, adjusted to pH7.3, was perfused for 30minutes followed by a rinse with 0.9% saline for
minutes. The animals were then divided into two groups.
After the rinsing rocedure the acidic decalcifyingagent New Decal3 (Bethelehm Trading Ltd,Gothenburg, Sweden) was administered through theperfusion apparatus (Hellstrtim and Bergh, 1984). NewDecalcR contains hydrochlonc acid 14%, PVP 7%, aquadest 79% and a trace surfactant. The decalcifying agentwas perfused at a rate of 6 mYmin and a hydrostatic