Decalcification by Per Fusion. a New Method for Rapid Softening of Temporal Bones

8/2/2019 Decalcification by Per Fusion. a New Method for Rapid Softening of Temporal Bones

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Histol Histopath (1991)6:415-420 Histology andHistopathology

Decalcification by perfusion. A new methodfor rapid softening of temporal bonesMagnus Nilsson, Sten Hellstrom and Nils AlbiinDepartment of Anatomy and Otolaryngology, Head and Neck Surgery, University of UmeA, UmeA, Sweden

Summary. 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.Key words: Decalcification, Perfusion, Immersion,Ear, Temporal bone, Rat

lntroductionThe 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 the specimens 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

Offprint requests to:Dr. Magnus Nilsson, Department of Anatomy,University of UmeA,S-901 87 UmeA, Sweden

causes 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)60 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 3%glutaraldehyde, 4% polyvinylpyrrolidone (PVP, m01 wt40,000) in 75 mM cacodylate buffer with 2 mM 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 5minutes. The animals were then divided into two groups.1) Decalcificationby perfusion (16 rats)

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


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Decalcification by perfusion

pressure of 120 cm H,O for 30 min (n = 3), 60 min(n = 3), 90 min (n = 3), 120 min (n = 5) and 240 min(n = 2). The perfusion with New DecalcR wasfollowed by a rinse with 0.9% saline for 5 min and theanimals were then decapitated. The heads were thenimmersed in the fixative salution for at least 48 hours.2) Decalcification by immersion (6 rats)

After fixation and rinsing by perfusion, the animalswere decapitated and the heads then immersed in NewDecalcR for 1 (n = 2), 2 (n = 2) or 3 (n = 2) days.The softness of the heads was checked daily by theneedle test (Preece, 1965). The solution was reneweddaily.After decalcification, al1 heads were dissected witha razor blade and the softness of the tissue wasevaluated. Sixteen left temporal bones were cut loosefor embedding in paraffin wax. From seven of theright temporal bones, specimens were collected fromthe middle ear for embedding in an Epoxy resin andtwo temporal bones were prepared for studies in thescanning electron microscope.The specimens collected for paraffin embeddingwere rinsed under tap water for 1 hour beforedehydration in graded series of ethanol. Thespecimens were then cleared in xylol for 30 minutesprior to infiltration and embedding in paraffin wax.The paraffin-embedded temporal bones were orientedand serially sectioned, 5 pm thick, in the frontalplane at 100 pm intervals. Also during the sectioningprocedure, the softness of the bony structureswas evaluated. From each time interval, sections

were stained with hematoxylin-eosin, van Giesonand periodic acid Schiff (PAS), examined andphotographed in an Olympus Vanox-S lightmicroscope.The specimens collected for plastic embedding, i.e.the round window niche, hypotympanon, fossa nasalisand tympanal orifice of the Eustachian tube, wererinsed in 0.1 M sodium-cacodylate buffer (pH 7.4) for30 minutes and postfixed in 1% osmium tetroxide inthe same buffer overnight. After rinsing in the samebuffer for 30 minutes, the specimens were dehydratedin a graded series of acetone, and embedded in anepoxy-resin. Thin (70 nm) and semithin (1 pm)sections were cut for electron and light microscopyrespectively on a Porter-Blum ultramicrotome(Instrument AB Lambda, Sweden). The semithinsections were stained with toluidine blue, examinedand photographed in an Olympus Vanox S lightmicroscope. The thin sections, contrasted with uranylacetate and lead citrate, were studied in a JEOL100XS transmission electron microscope.The two temporal bones for scanning electronmicroscopy were opened in the anteroposterior planeand the middle ear was divided into a lateral and amedia half.The specimens were dehydrated in an increasingseries of ethanol and then critical-point dried withliquid carbon dioxide in a Polaron E-3000 apparatus(Polaron Equipment Ltd, UK). The dried specimenswere then mounted on blocks and coated, undercontinuous rotation and tilting in a modified EdwardsVacuum Coating Unit E12E14 (Edwards HighVacuum, UK) at 1.3 kPa, with an approximately 20

Fig. 1A. Light micrograph (LM) ofthe rat Eustachian tube. Theepithelial lining with ciliated andsecretory cells (arrows) is wellpresewed, as are the tubalcartilage, adjacent muscles andglands. Parafin-embedded sec-tion, Hematoxylin-eosin. Peifu-sion-decalcified 120 minutes, x80. B. Detail of (A) at highermagnification x 340


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Table 1. Temporal bones decalcified with New DecalcR.

nm-thick layer of gold. The specimens were exam inedand photographed in an Cambridge S4 scanningelectron microscope (Cambridge Scientific InstrumentLtd, UK).The heads were considered sufficiently decalcifiedif they were soft and easy to dissect, the paraffin-embedded temporal bones could be sectioned withoutdamage to the specimen or the steel knife, and theplastic-embedded specimens could be sectioned in anultra-microtome without damage to either thespecimen or the glass knife (Table 1) .

30 (n = 6)60 .(n = 6)90 (n = 6)

120 (n = 4)240 (n = 4)

**

ResultsThe temporal bones from the rats perfused for 30or 60 minu tes with New D ecalcR as well as four o ut ofsix temporal bones perfused for 90 minutes were notsufficiently softened. The heads immersed for 1 and 2days were not softened (Table 1). Paraffin-embeddedspecimens from these groups could not be sectioned ina steel knife microtome without being distorted orcausing damage to the blade. Therefore the temporalbones decalcified by perfusion for 90 minutes or lesswere not processed further.The heads perfused 120 an d 240 minutes, as well asthe heads immersed for 3 days in New D ecalcR wereal1 softened. Al1 these temporal bones could be easilydissected, specimens collected and further processedfor examination in the light and electron microscopes(Table 1) .

ParatRn33342

The specimens were considered decalcified if 1) the heads were SOR and easy to dissect, 2)ttie parafin-em bedded specimens cauld be sectioned damage tothe specimen or the steel knife,3)the plastic-embedded specimens could be sectioned in an ultramicrotome without damage to the specimen or the glass knife.7.-PERFUSION Imin) "7.-INMMERSION

24h (n = 4)48h (n = 4)72h (n = 4)

Light microscopical findingsThe stainability of sections from the decalcifiedplastic- and paraffin-embedded specimens was goodand quite comparable to biopsies obtained from non-decalcified temporal bones (Albiin et al., 1986).The different kinds of tissues were generally wellpreserved. The epithelial linings as well as thesubepithelial connective tissue of the Eustachian tube

and the middle ear cavity had a normal appearance.

Plastio

32

2

Occasionally, small damaged areas were noted inwhich epithelial cells were lacking. Both ciliated andsecretory cells of the mucosa1 lining exhibited normalcharacteristics (Figs. 1, 2).Electron microscopical findings

TEM

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2

Scanning electron m icroscopic studies revealed tha tthe tympanic membrane (Fig. 3) lacked the rupturescomm only seen if not d ecalcified. The media1 wall ofthe m iddle ear also lacked fissures. Mucus covered thewalls, except for minor areas in which the epitheliumcould be studied. These epithelial surfaces exhibitednicely preserved ciliated and non-ciliated cells (Fig. 4).Furthermore, with this perfusion technique the vesselssuch as the stapedial artery were kept expandedthroughout the preparation of the specimen (Fig. 5).In the transmission electron microscope theepithelium of the middle ear showed minorultrastructural damage. However, it was generallyapparent that the perfusion-decalcified specirnens werebetter preserved than those decalcified by immersion.Cell organelles, e.g. mitochondna, showed ahomogeneous electron density in both the immersion-and perfusion-decalcified specimens. Widenedintercellular spaces and irregular-filled secretorygranulaes were only present in the specimensdecalcified by immersion (Figs. 6, 7).Discussion

SEM

2

2

Calcium carbonate and phosphate have to beremoved before bony specimens can be processed andsectioned with an ordinary microtome. The principalmethods to achieve decalcification includes treatmentwith either acid solutions or ion exchange resins.However, neither of these techniques is ideal andtreatment with acid for long periods of time invanablyproduces distortion of the tissue (Clark, 1954; Gussenand Donahue, 1965; Preece, 1965; Bussolati, 1978;Eggert and Germin, 1979; Kiviranta et al., 1980).

DecaOMed?

,No(6)No(4), Yes(2)

Yes(1O)Yes(4)No(4)No(4)Yes(4)


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Fig. 2.4. LM showing the round window niche. The epithelial lining changes from secretory and ciliated cells at the entrance of the nicheto a squamous epipithelium in the vicinity of the round window membrane (RW). SA = stapedial artery, ME = middle ear cavity, ST = scalatympani. Epon-embeddedd o n , oluidine blue, x 80. Perusion-decalcified 120 minutes.B. Detail of (A) at higher magnificatlon, x 340Flg. 3.Scanning electron (SEM) micrograph of the lateral wall of the middle ear with the tyrnpanic rnembrane FM) in its bony frarne. TheTM is sll intact but has lost its conical shape during preparation. x 60. Perfusion-decalcified 120 minutes.Flg. 4. SEM pictwe frorn the medial wall of the middle ear (fossa nasalis) showing squamous epithelial cells and a ciiiated cdl. x 6,000.Perfusion decalcified 120 minutes.Fi. 6. SEM picture of the stapedial artery, the stapes and the entrance of the round window niche. Note the bulgingarte which indicatesthat with tha pafusion pmcedure applied. the vessel lumen has erpanded throughout the preparation of the spedmen. ~e&on-decalc~ed120 minutes.


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le,

FFg. 6.TransmWoneiectron @EM)rnicrographofepitheliurn invicinfty af thetympg~alariRce fthe E m a ntube. Immembn-

~ t h l t ~iw-w fiM.

Fig. 7. TEM pictureof a similar area asin Fig. 7, but withthe specirnenperfusiondecalctfid for 120rninutes. Thestructure is betterpresenred andshows lessdamage than inspecimensdecalcifed byimrnersion.


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DiscussionCalcium carbonate and phosphate have to beremoved before bony specimens can be processed andsectioned with an ordinary microtome. The principalmethods to achieve decalcification includes treatmentwith either acid solutions or ion exchange resins.However, neither of these techniques is ideal andtreatment with acid for long periods of time invariablyproduces distortion of the tissue (Clark, 1954; Gussenand Donahue, 1965; Preece, 1965; Bussolati, 1978;Eggert and Germin, 1979; Kiviranta et al., 1980).The time needed for decalcication of osseousstructures is dependent on the minimum diffusingdistance of the specimen and the compactness ordensity of the bon e (Kiviranta e t al., 1980). Th etemporal bone is extremely hard. In the rat thepresent study showed that a period of up to three daysis needed to obtain a sufficiently deminerealizedtemporal bone when imrnersed in the commerciallyavailable New DecalcR. How ever, prolonged exposu reto t he acidic New D ecalcR causes digestion of cells,distortion of collagen fibres and poor histologicaldetail due to an impaired affinity of histologic stainsfor tissue structures (Gussen and Donahue, 1965;Eggert and Germain, 1979; Kiviranta et al., 1980;

Matthews and Mason, 1984).In attempts to obtain decalcified specimens byexposure to the decalcifying agent for shorter periodsof time, we have developed the perfusion technique.By perfusion, a fresh solution is constantly floodedthrough the tissue. This eliminates precipitation ofcalcium salts within the tissue and saturation of thedecalcifying solution by removed calcium, which o ftenoccur with the irnrnersion technique (Bussolati, 1978;Eggert and Germain, 1979). After the shortest timeneeded for adequate softening of the tissue, 120minutes, most structural details were well preservedat both the light microscopic and the electronmicroscopic level. Moreover, the specimens perfusedfor 240 minutes were equally well preserved, whereasthe specimens immersed in New DecalcR for 72 hoursexhibited artifacts, e.g. widened interceiiular spacesand generally po or histological detail , especially at theultrastructural level.Softening by perfusion with New DecalcR issuperior to the immersion technique in severa1 ways.It is not only faster but also preserves the lightmicroscopical morphology in a better way. Thetechnique is also suitable for electron microscopy.Further improvement of the perfusion technique is inprogress, e.g. by the use of other decalcifying

substances and in combination with microwaveirradiation (Book and Kok, 1987).Acknowledgements. This study was supported by grants frorn theSwedish Medical Research Council (17X-06578), the Ragnar andTorsten Soderbergs Foundation, the lngabritt and Ame LundbergFoundation, and the Medical Faculty, University of UrneA.

Albiin N. (1986). Middle ear mucosa in rats and hurnans. Ann.Otol. Rhinol. Laryngol. 95 (suppl 126), no 5, part 2.Anniko M. (1981). Effects of decalcification on the rnembranouslabyrinth. Micron 3, pp 267-278.

Baird I.L., Winborn W.B. and Bockrnan D.E. (1967). A techniqueof decalcification suited to electron rnicroscopy of tissuesclosely associated with bone. Anat. Rec. 159, pp 281 290.

Boon M.E. and Kok L.P. (1987). Microware cookbook ofpathology. Leyeden: Coulomb Press, Leyden. p 96.

Bussolati G.A. (1978). A fixation-decalcification procedure forbone biopsies. Histopathology 2, pp 329-334.

Clark P.G. (1965). A cornparison of decalcifying rnethods. Arn.J. Clin. Pathol. 24, pp 1113-1116.

Eggert F.M. and Germain J.P. (1979). Rapid derninerealizationin acidic buffers. Histochernistry 59, pp 215-224.

Gussen R. and Donahue D. (1965). Decalcification of temporalbones with tetrasodiurn edtate. Arch. Otolaryng. 82, pp110-114.

Hellstrorn S. and Bergh A. (1984). The use of an infusion set forgastric feeding as a perfusion apparatus in fixationdecreases the laboratoy hazards. Mikroskopie 41, pp 73-75.

Kiviranta l., Tarnrni M. and Jurvelin J. (1984). Fixation,decalcification, and tissue processing effects on articularcartilage proteoglycans. Histochernistry 80, 569-573.Kiviranta l., Tarnrni M. and Lappalainen R. (1980). The rate ofcalciurn extraction during EDTA decalcification from thinbone slices as assessed with atornic absorptionspectrophotometry. Histochernistry 68, pp 119-127.

Matthews J.B. and Mason G.I. (1984). lnfluence of decalciyingagents of irnrnunoreactivity of formalin-fixed, paraffin-ernbedded tissue. Histochernical Joumal 16, pp 771-787.

McDonald D.M. and Lame D.T. (1983). The ultrastructure andconnections of blood vessels supplying the rat carotid bodyand carotid sinus. J. Neurocytol. 12, p 119.

Pearse A.G.E. (1968). Histochernistry, theoretical and applied,3rd ed, volurne 1 :J&A Churchill Ltd. London pp 579-581.

Preece A.A. (1965). A manual for histologic technicians, 2nded. J&A Churchill Ltd. London. pp 103-108.

Accepted March 1, 1991

Decalcification by Per Fusion. a New Method for Rapid Softening of Temporal Bones

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