Dr. H. H. Lindeman E. N. T. Department · Ullevr.illlospital OslofNotway ISBN 978-3-540-04464-2 ISBN 978-3-642-99960-4 (eBook) DOI 10.1007/978-3-642-99960-4 Das Werk lst urheberrechtltch geschO.ht. Die dadurch begl1indeten Itechte, lnsbcsoildere die der Obersctznng. f.lcs Naehdruckes, der ·gutnahme vou Abbildungen, der }!'unksnndun{!, dcr Wiedcrga.be aof pbotomechanisch~tn oder iihntichem Wege und der Spe.icberung in Datenverarbeitungsanl~en blelbcn, aueh bei nur atiMwgswclser Verwertung, vorbehalten BeJ VervielfAltigungen tor gewerbliche Zwecke ist gemAB § M UrhG eine Vergiltung an den Verlag zu zahlen. deren HOhe mit dem Vcrla.g zu verelnbaren ist © by Springer-Verlag Berlin Heidelberg 1969 Originally published by Sprlnger-Verlag Berlin Heldelberg New York in 1969. Softcover reprint of the hardcover 1st edition 1969 Library of Congress Catalog Card Number 6fJ·205S'2. in 1969 Tltei·Nr. 6961. Die Wkldergabe von Oebrauchsnamen, Handelsr.amen, Warenbezcichnungcn usw. In diescr Zeltschrift berechtlgt. aueh ohne bosondere Kenoz:efchnung nfcht zu der Annahme, daD solehe Nn.rncn im Sinne dcr Warcnzeiehen-und Markenschutz-Gesetzgebung als frei zu betrachten wA.ren und dn.her von jedcrmann bemrtzt werden dUrften Contents I. Introduction. . . . . . . . . 7 II. Material and Methods. . . . . 8 A. Preparation, Fixation and Staining H B. Microdissection for Study of the Gross Anatomy of the Vestibular Lahyrinth !l C. Microdi",.ection of the Vestihular Hen"ory Regions !J D.Light and Electron Microscopy of ~ection" . . . . J() E. Discussion . . . . . . . . . . . . . . . . . . II HI. Form and Interconnections of the Vestibular Duets and Hacs 13 A. Introduction . . . . . . . 1:3 13. Material and Methods . . . . . . . . 14 C. Observations and Comments . 14 IV. The Vestibular Nerve and Its Ramifications 24 A. Introduction . . . . 24 B. Material and Methods . . . . 25 C. Observations . . . . . . . . 21i I. General Course of the Peripheral Fibre;; of the Vestibular Nerve 2fi 2. (',ourse of the Myelinated Xen'e }<'ihres to the ~laeula utriculi. 2fl 3. Course of the Myelinated Nerve Fibre" to the ~Iaellla '''('culi 26 D. Discussion . . . . . . . . . . . . . 2S V. Structure of the Vestibular Sensory Epithelia 31 A. Introduction . . . . :11 B. Material and Methods . . . . . . . . . :12 C. Observations . . . . . . . . . . . . . :15 1. General Structure of the Sen"ory Epithelium. 3il 2. Macula utriculi ........... . :1\1 3. Macula sacculi . . . . . . . . . . . . . 46 4. Sensory Epithelia on the Crist.ae ampullares 4H D. Discussion . . . . . . . . . . . . . . . 52 VI. Morphological Polarization of the Sensory)jells £;7 A. Introduction . . . . 57 B. Material and Methods 51{ C. Ob$;ervations . . 58 1. Macula utriculi . . 58 2. Maeu1a sacculi . . li2 3. Sensory Epithelia on the Crietae ampllilarcs fi2 D. Discussion . . . . . . . . . . . . . . r,4 VII. Innervation oLthe Vestibular Sensory Epithelia 71 A. Introduction . . . . 71 B. Material and Methods . . . . . . . . . . 73 C. Observations . . . . . . . . . . . . . . 73 1. Innervation of the Sensory Epithelium on the Cristae ampullare. 73 2. Innervation of the lIIacul" utriculi 77 3. Innervation of the l\Ja.cuIa sacculi 78 D. Discussion . . . . . . . . . . . . 78 6 Contons VIII. Form and Structure of the Statoconial Membranes and the Cupulae 83 A. Introduction. . . . . 83 B. Material and Methods. . . . . . . . . . . . 8:1 C. Observations. . . . . . . . . . . . . . . . 84 1. StatoconialMembrane of the Macula utriculi 84 2. Statoconial1l1embrane of the Macula sacculi • 88 3. Cupulae 90 D. Djscussion. . . 92 IX. General Discu88ion 96 X. Summary 100 Acknowledgement. 102 References .. 103 Subject Index III I. Introduction The membranous labyrinth lies ('neloHed in the very hard petroua part, of the temporal bone and, as the name implies. it,s structure is extremely compl('x. This may explain why our knowledge of this organ and the vestibular sensory regions within it, is not yet satisfactory. In recent years the rapid development of space research has provided a power ful stimulus to our interest in the vestibular apparatus. This has found expression in annual symposia, in which the role of thc vestibular organs in the exploration of space is discussed. However, little is known as yet about the inflU<'nce upon the equilibrial apparatus, during space flight, of weightlessn('ss and other relatpd conditions. The inner ear has also acquired increased significance from an otosurgieal point of view. Operations are today performed in regions pnwiously inacecSHibl" to surgery. This requires exact knowledge of anatomical details and of ['{'Iations between the different st,ructures in the inner ear. However, the majority of illustrations available on the morphology of tlw vestibular apparatus are of little value for preRcnt day surgery or cxpl'rimcntal investigations. The main reason for this is undou btly the limitations of the histo logical methods used. Most studies on the Yestibular sensory regions ha,'!' hCl'n carried out on serial sections of deealeified and embedded temporal hon('s. i-linee usually a limited number of sections perpendicular to the epithelium are suitahlc for closer study, one can only stud~' small areas of the vestihular sensory fPgiolls in each temporal bone. Furthermore, decalcification and emhedding are time consuming procedures and they arc the cause of frequent artefacts, often mis interpreted as pathological changes. Orientat,ion, a necessary prerequisik whpn a comparison of the same region in difterent animals is attemptcd. is likewi;;e difficult in such sections, and the rolat,ion of certain cells to others is also lost. Even though electron microscopical studi!'s have shown that the st fudUl'C of the vestibular sensory epithelium is cOIlHidern hly more COIl! plieat.erl t han wa,~ previously realized, such investigations can only be used to a limite'l ('xt.·nt in quantitative studies. :Furthermore, only smull areas are illvest,igatcd hy t/W8C methods, and the loss of orientation inevita hIe in studies under high-power magni fication makes i1l difficult to carry out systematic investigation of ,[dini!e areas. This is probably the reason why it has not been possible to follow up satisfactorily the study of regional differences in the structure of the sensory regions, described primarily by Lorente de N6 (1926) and Werner (1933, 1940). Differentiation of the sensory epithelium is seldom discussed, and it it still usual to regard the maculae as "flat spots" of uniform structure. 8 H. H. Lindeman: The present study is based on new methods of microdissection of the mem branous labyrinth and the vestibular Hensory epithelium. Using primarily these methods, an attempt has been made to: a) Demonstrate the gross anatomy of the membranous lab~Tinth and the peripheral branches of the vestibular nerve. b) Assess the form and size of the vest,ibular sensory regions. c) Investigate whether there is a special pattern in the vestibular sensory regions regarding the structure and innervation of the sensory epithelium and the structure of the statoconial membranes over the maculae. d) Carry out a quantitative estimation of the number and distribution of the two types of sensory cells. For practical reasons, some of the investigations were made on only one species of animal. The temporal bone of the guinea pig, which forms the basis for all the studies, was used in these cases. To facilitate reading, a comprehensive account of material and methods is given separately (p. 8). In addition, a short review of the material and methods used in the special investigations is given in each chapter, which also includes a short discussion of the findings. A general discussion is found on p. 961. II. Material and Methods Approximately 200 guinea pigs, non albinos, weighing from 2;>0 to 350 g were used in the investigation. All of them showed an obvious Preyer's reflex (twitch_ing of the pinna to a sound stimulus). These guinea pigs made up the bulk of the material. In addition the temporal bones from n rabhits, :J catR. 4 Rquirrel monkeys. 4- humnn f(){~tuse8 and 4 human adults were included in the study. The temporal bones from both sides were used. A. Prcparntioll, Fix:ttion and Staining In most cases the instruments and techniques described by Engstrom et al. (1966 b) were used to prepare the temporal bone for fixation and staining. Only the most important features will be mentioned. 'fhe guinea pigs were decapitated without an ..... sthesia, the other experimental animnls under sodium pentobarhital anaesthesia. The lower jaw was removed, the bullae tympanicae exposed and OI)('1w<J. After making a wide optming to the vestibule from the Im",,,l part of the rod.l.a, fixing fluid was injected int" the vestibule, and the specimf'" was then placed in the fixntiv('. 1n t.he g1linea. pig it took 1--2 rninllt-cR bdore hot.h temporal honCR were expoHed to the fixing fluid. In the rahbit, cat and monkey, where the bone iR stronger, the same proccdurn took 2-5 minutes. The following fixatives and staining procedures were used: 1.0.,mium Tetroxide (ORO,). Most of the material was fixed and stained in a O.5--J.5% solution of vcronal-huffercd osmium tetroxide for I-a hours in 11 refrigerator at ahout, 4° C. After washing in physiological saline, the temporal bones were ready for further preparation. If they were not used the same day, they were transferred to 70 % alcohol and stored in a refrigerator. 2. ]fIethanol/Ether Fixation ana Siaining with Giemsa Solution. After fixing for at least 2 hours in equal quantities of methanol and ether. some temporal bones were stained for abollt 5 minutes in concentrated Giemsa solution. They were then transferred to xylol for further preparation. l. Some preliminary results of the present study have been published previously by Engstrom et al. (1966a), Lindeman (1967). Morphology of \,estil}lliar S"n.ory Reginns 3. SiltH3r Nitrate. \Vithout previom; fixation th(, Rf'IlS0fY ff,).!iom; in Rome animalfol W(>T(, isolat-ed in Hinger's solution and tram~fefJ"ed' to O.r.u;~ Agi\03 for ahout r; miullt('R. Aft(>r rinsing in distilled water, the Rpedmens were expoRe<l to ultmviolet light IIntil they Required a dark colour, hy precipitation of silver chlorid(~ (Ree HOHlPis. 1H 4S). Furthpr prnparatif)n wa.~ carried out in xylol, after dehydration, 4. Fornutlin. A small number of temporal borl£'S Wf'rp fixed in Ino~ formalin and prepllrNI without staining. ,5. Formalin Fixation a:Jui Btaining with Sudun Blflfk IJ, according to the method dNwrihed by Rasmussen (19rH), was IIsed in some eRRes. 6. Modified Maillet's (19(j3) Method. This was IIHed in a great nllmher of animals to .tlldv t,he innervation of the sensory epithelium. The fixing/I.;taining solution eonRish·d of a mixhl~t" of 1.5% osmium tetroxide Rotution and zinc iodide di~HOlvt"d in dilitilled wah'r (A('(' t:ngRtri)1lI ef al., 1966b). All the temporal bones from humans. both .duits and foet"RPR. wen' fixed by Dr. ilrl'(U",rg in 1.5% osmillm j"troxide soilltion (see Bredberg. 19n5). The vestihular lahyrinth was diK. sected by using the same technique as in animals. B. lmcrodis~('ction for Study of the Gross Anatomy of the Vm;tibular Labyrint.h These inv£'Rtigations were carried out on hoth dl'('aleifiml and non-de('akified ~JI('einwns. A number of temporal bones from guim·(t pi~H and ra"hit~ w('re c)N·al/·ifil·d in ;;";, H:\()3 until it was possible to remove the bone 8ubstam'{' with forccps. Jil tlH' guif"'" pig about 4 hours, and in the rabbit about 20 hours, Wf're found to he Rati!'tfa(·toT~·. Aftt'T m'\l1 raJi7.ing in ,:Na2S04 the specimens were rinsed in watf'r. Mierodisst',,·tion wa~ ('arri{'d out Imd('r the ~tt'rpo· microscope either in distilled water or, aftprdehydratioll. in xylo1. In xylol thn hom-HtrtH·ture is translucent. but the membranous labyrinth is ali-'o f'O tranRparcnt that it iF!, Nt.-;il.v damagNI during preparation. By making the eli'Section in distilled water it was po,"ihl" to isolate almost the whole of the memhranous lah,'rinth (Yi~. I). In ordrr to study smali parts of it in detail, these were detadlCd, mOllnh-d in Canada t);d.r.mm or ~lv{'('rirl on a S/if/(·. {·O\Trf·d with a cover glass and Rtudied und('r an onlitlury light mkroR(,op(': The general st.ructure of the labyrinth was also ~t IIdied in non-d('udf·ifif'd h·mporal bonf's from all species. Furthermore, a good view of th(' anatomy of the hbyrinth waA obta;'",d when the vestibular sensory regions were disRcet.ed fOT det.ailed !'ltlloy, C. l\1icrodisst~cti()n of the Vestihular St'lISOI'.v R('~if)ns Dissection was carried out. on non·dc('aldfipd h'IIlporal h()I1(,~ IIndt'T th(· Rtef(·o1lli('f(j~(·op(,. Using powerfnl hooks and watchmaker', foreeps. "nd ROJlwtillll'S " dmtal drill a. '1''''1. tlw facial canal wa. opened and the fa<'inl IH''''" rentov,,". The lateral wall of tlIP vestihule to· ~cther with parts of the walls of the OHRNlUR Illllpllllae wrrl' Hu'n rrft)o\·('(1. thus rXl'oRiflg the ~m(~etll('. th(~ utride a,nd the HtTP(' Itwmhran()l1~ arnpuJl;w (Fig-. 2). Thil'\ pro(',plillr(' took only a eotlpl{' of minut.es in the gllinea pig. hut, filightl,v longpr ill the o1iH'r RI)('('j(,JoI wlll're tlU' bone waR hllnltlr. As mentioned previously, a good idt':a of the anatomy of the mClllhraOOltK labyrinth wa~ ohtained during the dh~Rcetion. "More pronoun(·(·d pathologipal conditions could alHo bp !Wf'n with low power magnification (p. 55). Further dissection was carried out. IInder X 411 magnification with fim' \\'atehmaker's forceps. The saccul", was opened first. Gentle "quirts of fluid through a thin pipctt., were usually sufficient to separate the "Intoconial memhrane completely from the ,,'nsory epi. thelium. Oecasionally a pair of forceps was ueeded to help to free it. The Rtataconial m!'mhranc was t,hen placed. under the fluid. on a cover gla8~ which was lifted c<l.refully ont of t.he fluid and placed on a slide. It could now be studied mol'~ closely under a light. and phase. contrast microscope, and X.rays could be taken (see p. 85). The sen~ory epitheHum was t.hen st'parated from the sl1bt'pithdiaJ tiRRtle. A very sharp knife was used for this purpose. The knife was introdurM under the sen!l:ory epithelium 10 H. H. Lindeman: outside its outer limits. It was easy to loosen large areas of the sensory epithelium in osmium· fixed preparations if one entered at the correct level. If the knife was introduced carefully under the sensory epithelium, it was possible to remove it almost completely in most cases. It was next transferred to a drop of glycerin on a slide, with the upper surface of the epi. thelium uppermost. This surface specimen was then covered carefully with a cover gla88, reBdy for further study by light and phase.contmst microscopy. Epithelium that had not heen fix~d in osmium tetroxide was more difficult to separate from the underlying ti88ue and could UWFl,foFl' usually Ix, studied only in fl·ngmont •. The macula utriculi was then prepared in the same way. In this case, however. the whole sensory region was released from it. attachment anterior to the bone. It Was not as easy to detach this sensory epithelium as that of the macula sacculi, because it was often difficult to fix the objcct during di88cction. If. however, a good hold was ohtained in the subepithelial tissue with forcocp., it w". Marly always posHible to iHOlate the sensory epithelium alrneHt. completely and to tmnsfer it to a slide for further study. Finally the ampullae were dissected free, first the lateral then usually the anterior and finally the posterior. The roof and lateral walls of each ampulla were removed 110 that the crista ampullaris could be exposed. The cupula was removed and mounted on a slide for further study. The sensory epithelium was then separated in the same way as that of the maculae. By holding the specimen by the ampullar nerve, good fixation was obtained, and it was relatively easy to free the sensory epithelium in osmium·fixed preparations. The epi. thelium, which normally follows the saddle shape of the crista. is flattened once the cover glass is in the place. Light and phase.contrast microscopy were carried out with a Wild M. 20 Research microscope. The sensory epithelium was viewed from above. Low power magnification (x 60-100) was used to observe the form of the sensory epithelium. and certain structural peculiarities were a]so then apparent. Higher magnification (x500-1,000) was used for detailed study. By focu88ing at different levels it was possible to obtain optical sections through, for example, the hairs of the senllOry cells, the surface of the epithelium and the nuclei of both sensory cells and supporting cells. FocuBBed at a given level, the structures could be seen clearly without troublesome interference from structures at other levels. By moving the specimen. different regions entered the field of vision. In this way the whole stnlcture of the .cnROry epithelium could be investigated rapidly, and the different regions rel"ted to one another. Blood veSBels and nerve fibres in the subepithelial tissue of the macu lae were also studied directly after di88ection. In the saccule this ti88ue, lying in a groove in the bone, was easily prepared. The subepithelial ti88ue of the macula utriculi was, as mentioned above, released before dissection of the sensory epithelium and could also be examined. So also could the subepithelial ti88ue be studied with the sensory epithelium intact over it. Although these specimens were often so thick and dark that detailed study was difficult, it was possible to correlate individual intraepithelial areas with corresponding subepithelial regions by focu88ing at different levels. When it was desired to study other regions of the membranous labyrinth, these could be di88ected free for direct study or embedded for section later. D. Light and Electron Microscopy of Sections Temporal bones from guinea pigs and squirrel monkeys were used for this purpose. They were fixed in cold 1.5% veronal·buffered osmium tetroxide, and stored for 1'/. hours in " refrigerator. After washing in Ringer'. solution, they were dehydrated in iner"",,;ng conc.m trations of alcohol. The vestibular sensory regions were dissected free in absolute alcohol, transferred to propylene oxide and embedded in Epou. Sections for Iight/phase.contrast aud electron microscopy were carried out with a LKB Ultrotome. Sections for Iight/phase.contrast microscopy were staine4 with pamphcnylene diaminc, sections for electron microscopy with lead acetate, lead citrate or uranyl acetate. Electron microscopy was carried out with • Siemens Elmiskop I A. Morphology of Vp ,Rtibular Sensory R~gion., II Macroscopic pictures of the labyrinth wern taken with ft roll film ('<tllH'Ta, HaRHPlhla.cJ 500 C. equipped with a Zeiss S·Planar 120 mm lens. with exlen,ion tubes of varying I"ngth (2.1-150.0 ern). The microscopic pictures were taken under a Wild }[. 20 Research miero· scope. Kodak Tri·X Pan 120 film was used. E. Discussion In the last ccntury, anatomists relied t.o It large ('xt.ent upon dlssect.lOn for studying the structure of the inner ear. The most outstanuing work of that period is Retzius' "Das Gehiirorgan der \Virbelthiere" (188Ia, 1884). Retzius combined the study of sections with microdissection of specimens fixed chiefly in osmie acid. Microuisscction was of greatest value for investigating the gros.~ anatomy of the labyrinth, but it was also used for dctailCld st.udy of HlC ('oehlear and vestibular sensory regions and other parts of the membranous labyrinth. Retzius' plates thus show preparations of the vestibular sensory epithelium - where the pattern of sensory and supporting cells appears - from the maeula sacculi of the alligator, from the macula utriculi of the pigeon and from the cristae of the rabbit and the cat. Microdissection of the temporal bone was also used to Rome extent, for ana· tomical studies of the labyrinth, in the first half of the present century (for example Kolmer, 1927; de Burlet and Hoffmann, 1929; Werner, 1940), but not for direct study of the structure of the sensory epithelium. Similarly, in the last 'decade, microdissection under the st€reomicroscope of parts of the membr~nous labyrinth, for embedding for electron microscopy, has given a good idea of the anatomy of the inner ear. . More recently the microdissection method has been resumed for im'estigation of the organ of Corti (Neubert, 1950, 1952, and others). In particular Engstrom and his group (Engstrom et al., l!162; Engstrom et al., 1966b; Kohonen, Hl65; Bredberg et al., 1965; Bredberg, 19G8), applying a sp""ial technique, have carried out systematic studies of the organ of Corti. Vsing osmium tetroxide aIHI Jwrve· stained specimens, they have demonstrated the advantages of the method, hoth in experimental animals and in man, for the study of normal and pathological epithelium and for quantitative analysis of tIl(> cochlea. This type of systematic study of surface prc-parations has apparently not he!'n carried out in the vestibular sensory regions. Xeumann and Xeubert (1958), how· ever, released the macula utriculi with its subepithelial tis;;ue and studipd the epithelium directly under the light microscope. However, probably because of the thickness of the specimen and the techniques used in fixation and staining, these studies provided no information ahout cell patterns, nor about the hairs of the sensory cells and other structural details. r~ngstrom et al. (1962) and Ados aud Engstrom (1965) used microdissection for studies of both fixed and non·fixed vestibular sensory epithelium. However, .Tohnsson and Hawkins (1967), who used osmium tetroxide for fixing, considered that the maculae and cristae were t.oo dark for surface studies. The technique described in the present study for microdissection of the vcstib· ular sensory epithelium should provide a useful supplement to the study of sections. The instruments required arc few and inexpensive. \Vit.h a little training, the investigator may find the vestibular sensory regions rapidly and easily, 12 H. JI: Lindeman: especially in the guinea pig and the human foetus, but the procedure is rather time-consuming in the adult human temporal bone. A good view of the anatomy of the labyrinth is obtained during dissection. Separation of the sensory epi thelium from the subepithelial tissue needs training and some care, but it is sur prisingly easy in osmium-fixed preparations. It is possible, for example, only a couple of hours after decapitating a guinea pig, to have the sensory epithelium isolated from all the cristae and maculae of one ear, for light and phase-contrast microscopy. The long waiting period and the artefacts which arise as a result_of decalcification and embedding are avoided. Almost all the sensory and supporting cells in any sensory regions can be observed. These cells can be related to one another, and to other regions and other structures. Orientation in the specimen is certain, a factor of decisive importance when it is desired to compare corre sponding regions in different animals. For these reasons, the method seems also to be especially suitable for quantitative assessment of both normal and patho logical epithelia. When desirable, other parts of the membranous labyrinth can be dissected in the same way as the cochlear structures (Engstrom et al., 1966b), for direct study or for embedding and sectioning. Osmium tetroxide, which was used in most of the studies in the present in vestigation, is the most common fixative used in electron microscopy of the struc tures of the inner ear. In addition to providing good fixation, it has several other advantages; the membranous labyrinth is seen very distinctly under the stereo microscope; the sensory epithelium is easy to separate; and-it is possible to study the hairs, the epithelial surface and the intraepithelial structures in the same specimen and under high magnification. If it is not desired to study the specimens at once, they may be stored in 70% alcohol in a refrigerator Storing for 1-3 days in alcohol does not seem to have any significant effect on the epithelial structure. Moreover, the sensory epithelium is easier to free, undamaged, from the underlying tissue. If, however, preparation is postponed for a longer time, the epithelium becomes so adherent to the sub epithelial tissue that itmay be difficult to separate it as an entity. It also becomes darker and the cell pattern is seen less distinctly in phase-contrast microscopy. Especially in the guinea pig where the crista has the shape of a fairly sharp ridge, the epithelium may be difficult to flatten, it often folds together and it is less easy to study. Sometimes it is difficult to Bee the cell pattern because of numerous granules in the epithelium. These are most obvious when the specimen has been stored for a long time in osmium tetroxide or in alcohol. It is often better t~ fix for shorter periods or in concentrations of osmium tetroxide solution lower than 1.5%. Distortion of the sensory epithelium occurs occasionally. This is often due to the epithelium having been mounted in too little glycerin, and occurs most easily in specimens fixed for a short period. Distortion can, however, be recognized by a change in the cell pattern at the level of the epithelial surface, and by folding down of the hairs of the sensory epithelium. This type of surface specimen is not suitable for assessment of the area of the sensory region nor for estimation of cell density in different regions. The specimen can, however, be used for some structural studies and for estimation of, for example, the relationship between the numbers