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Studies of ion distribution in the inner ear: scanning electron microscopy and x-ray microanalysis of freeze dried cochlear specimens
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elect~on im were c~mbined with energy d~spcrsi,~e ribuflon in z,Mried~ir~ch~ inner ears. Fluid residues ~ of mOteristic~lly shaped crys-::~ ~~d from Crys~ls ~m consistem-withthe ~ .In , um lov©lsin perflymph and sodi: ~&at~ ty of the major fluid comp~.rtments
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,~ e~n~y inenti,-~ ,o those ob~inocl from ¢ochle~ :in ionic compo~flon of e~dolymph in the two divisions of the of the organ of Corti does not form crystals, w~ch suggests that the ~ ph or endolymph. Secondly scanning electron n of c~L~membrancsof most cochtea~ tissues, features of t'~ue0 such as interior cell ~tered scanning electron ima~dng was ' the identification of crystai~ of fluid
two major fluid ad ~v¢~al miall~r spaces nments. In addition, the ays ~ impoxtant role in ,
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that endolymph ir~s variety of species contains approximately 150 mEquiv./l of potassiu m and I l0 mEquiv./l of chlorine. However, there has been fa~ less agreement concerning t~he level of scdiura in this fluid, with values ranging from 1.0 to 1i5 mEquiv./l [1,7,12, 14,!8,24]. It seems likely that the lowest measurengmts of closer to t:le ~ctual level. Higher levels presumably reflect the uncontan~n~ted sample of endolymph with the invasive techniques currently employed. Both perfiymph and endoIymph contain about 6 mEq~v.]l of calcium, and traces of other inorg~c ions [3,14,18]. Protein concent~tion of the two fluids has ~ measured at 1-2% of the level in senan, with perilymph c o n ~ more protein than ~ d o lymph [2,16,23]. However, vital staining of the cochlea has ~oduced e v i ~ that a glycoprotein ~latfix may be suspended wihhm the endolymphafic ~ace [2,5]. It has been su~ested on s~atomical grounds that endolymph in the utricle and cochlea may circulate in.dependently [20-22]. In support of this b y , thesis, Rodgers and Chou [18] reported that sodiv.m levels in scala media are higher than those in the utride. Conversely, Sellick a~.d John~tone [21 ] concluded in a renew of several st~Jdiesthat sodium levels are higher in vestibular eadolymph than in cochlear endotymph. The ionic composition of the fluids filling |he various subcompartments of the cochlea - such as the inner sulcus, the subtectoriat ~pace and the extracellular spaces of the organ of Corti - have not been established with certainty. Anatomi~ data obtatined by Engstrom ~5] led him to suggest that organ of Corti fluid spaces are filled with a fired with an electro|y~:e composition similar to perilymph, but with a significantly ~ r prorein concentr~fion. He termed this fluid 'cortflymph'. The fluid filling the subtectorial space and the inner sulcus has been variously identified as endolymphati¢ or peri. lymphatic in composition, based on anatomical [I 1,19], physiological [9,10] and X-ray microanalytic [6,19] grounds. The intraceUular ionic composition of hair cells and stria vascularls cells have not been directly assessed. However, given our current understanding of intrscellul~ environments, it seems likely that potassium, chlorine and organic cations are the dominant electrolytes. The major difficulty inherent in the measurement of the ionic composition of fluids in small extracel~ular compartments, and even in some relatively large extraceHular compa~ments, is fluid contamination from one compartment to another during ~ p l i n g . The wide range of values of sodium content measured in samples of endolymph [ 1,2,7,12-~14, 18,23,24] attests to the seriousness of tt'ds problem. Since fluid sample collection necessi. tares the violation of fluid space integrity, techniques have concentrated upon minimizing opportunities for ion movement. The use of glass micropipettes has been the ~ d ~ d mode of obtai:ning fluid samples, but the success of this method is quite v~able. $in~at objections can be made concerning ion.sensitive microelectrodes. Oliviera et al. [13] employed cryogenic freezing of the cochlea and dissection of fluid samples in a frozen. hydrated state. However, sodium levels obsorved in endolymph with this technique were about 40 mEquiv./l, again suggesting contamination. Cryogenic fr~ezing followed by freeze-drying is commonly employed to immobilize " 'o n of freeze-dried"tautenS" l ~ the ohions in complex biologic specimens [17]. Di~ct~: vious advantage that sampling is performed in an anhydrous state, e l ~ a f i n g ion movement due to fluid displacement or to diffusiono However, the bony capsule of ~he e ~ e a necessitates freezing of the inner e~ intact. Whether such freezing in fact preserves the
integrity of e~hlear fluid compartments is not clear, especially given the data of Oliviera et el. [13]. The size of the intact cochlea necessitates freeze-drying at moderately low temperatures (--40 o C). !t has been demonstrated that such temperatures allow some movement of ions during the freeze-drying process [4]. Whether movement of ions during not known. Wtfil~ some freeze
METHODS
Normal chinchillas (Chinchilla laniger) were mesthetized with sodium pentobarbitol (?0 mg/kg) and ketamine an~ the cochleas rapidly dis~cted from the each subject:- was taken within 60-90 s, and second within 120-180 s, of decapitation. Care was taken not to breach the round and oval windows during dissection. The whole cochlea.s were then quenched in Freon 12 cooled to its liquid-solid interface (-159°C) ~ d freeze-dried at 0.01 Torr and -40"C for at least 72 h. After freeze.dzying, specimens were maintained under vacuum over calcium sulfate. All samples were di~cted with instruments which had been washed with #ass-distilled water followed by anhydrous acetone, Specimens for ~ e present inv~stigation were obtained from the upper basal and lower second cochlear turns of the cochlea, ,rod the utricle. The bony capsule of tbe inner ear was fractured, beginning at the medial margin of the round window, opposite the ovfl window. The bone was then pid,:ed away with fine scalae with forceps and probes. ,,ments. The free juaction of o,g ofco,ti ofte be further the outer ~lls ~,outer hai~: d ~ c t e d by ~p~ati ~gments ~ticells w i ~ or wi~ou l ~ g the methods ( ~ spiral i:iga. ment be di~eted as a U~t, or stria could hi; ~p~atea tram the un~eriying ligament by ~ r t i n g a razor-blade fr and twistir g, which caused 1:he two tissues to separate naturally as de~ribed by Tlalmarm et el. [25 ]. Outer h ~ cells from the second turns could not be isolated under light mi :roseopic v i s u ~ t i o n . However, when lon~tudinal fragments of the ~rgan of Corti were identified ruder ~ing electron micro~ope as having only outer hmr cells, them fra~ents could later be removed from the SEM stub md tea~d apart to obtain mdivid-
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Fig. I. EDXA spectra of N ~ l and KCI controls° EDXA spectra obtained from crystals of t~ze-dried physiologic salt solutions, a. Reagent-f,rade NaC|. The spectrum reflects the differential emission of X-rays from sodium as compared to chtorine, Approx~ately four times as many emissions are observed in the chlorine window as are seen in the sodium window, when the two eleme~its a ~ pre~nt in equal proportions, b. Re~ent-grade KCI. Cldorine emits about 20% more X-rays than potassium, when the levels of the two elements in the excited c r y s ~ are the same,
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All specimens were mounted on carbon planchets with plastic cenent. They were then coated with carbon. Carbon coating gives secondary scanning images similar to those seen with heavy metal coating, but does not interfere with backscattered imaging or EDXA as does the metal coating. Imaging of specimens was performed an an E I'EC Auto~can SEM. Instrument parameters included an accelerating voltage of 20 keV, ,::ondensor current of 2.5 A and working distance of' 16 mm for secondary ima~ng, and a condenser current of 1.8 A and working distance of 13 mm for backscattered imaging. 'i~le ETEC Autoscan was equipped with a KEVEX Quantex system for EDXA. A conden~or current of 2.0 A, working distance of 13 rnm and tilt of 45 °, was employed for all EDXA measurements. Specimen current varied from 0.25 to 0.75 hA. Dead times were liraited to between 30 and 70%. Initial work demonstrated that we routinely could exp~;ct K~ peaks in the 1.01-1.09 keV (Na), 1.98-2.06 keV (P), 2.27-2.35 keV (S), 2.59-2.67 (CI), 3.283.36 keV (K) and 3.66-3.74 keV (Ca) windows. Both a matrix corre~-tion factor and correction against an adjacent r e , o n of carbon and glue were used to e iminate background emission initially, but the measured background was ~,~eleetedfor routine use. EDXA dala were coflected at a magnification of 15 • 10a from 0.5-2.0 tam crystals of perilymphatic or endolymphatic origin. The emission of X-rays, measured as counts/s or as peak.to-backgz ound ratio, does not provide an absolute measure of element abundance. The atomic nu~ber of an individual element, interactive effects between different element:~, specimen de~sity and other operating factors can influence tke number of emitted X-rays which are d.te~ted from a given specimen. To provide a simple standard for comparison with cochlear fluid residues, droplets of 0.9% solution of reagent-grade NaCI and KCI were frozen ant freez'-dried on carbon stubs, and prepared for EDXA in the same manner as cocMea~ specimens. Typical EDXA spectra obtained from the resultant crystals are shown in Fig. 1. These spectra illustrate the differential emission of X-rays from the different eleme~ats which are of partitular importance as electrolytes in the inner ear. '
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RESULTS
The fluid resid~.les which remained after freeze-drying differed with their location. Crystals in a tenuous matrix were found in isolated clumps within the perilymvaatic spaces of scala tympani and vestibuli. The crystals varied widely in size and were roughly cuboidal in shape. Fig. 2 illustrates isolated perilymph residue, with an associated EDXA spectrum. The spectrum indicates approximately equal peak-to.background levels of sodium and chlorine, with small but measurable quantities of potassium and calcium. Fluid residue in the scala media frequeltly formed a cottony mass which filled the scala. Under SEM, this residue was similar in appearance to the perilymph residue, except that the crystals tended to be slightly elongated. Fig. 3 shows fluid residue on the upper and lower surfaces of the rectorial membrane, with associated EDXA spectra. Both spectra display a relative excess of potassium e~er chlorine, and an extremely low level of sodium.
Fig. 4 ~ustrates isolated fluid residue and otocon~a from the utficle. EDXA spectra obtained from an otoconial crystal and an endolymph crystal are also shown. As illustrated in the figure, endolymph spectra obtained from the utricle are not noticeably dii-
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Fig. 2. Freeze-dried perilymph residue. Secondary and backscattered electron images el clumped perilymph crystals, isolated from u:ala Wmpani of the freeze-dried inner ear. The crystals are bour~d together by small amounts of an ~tmorphous matrix. The EDXA spectrum was obtained from the small crystal h~dicated by the arrow, the larger masses being inappropriate for EDXA due to suppressed emission of X-rays from sodium. The inner border of the box surrounding the crystal in the seco~,~dary electron image represents the region excited hy the electron beam during EDXA. The perllymph '~pectrum i:~ essentially identical to the NaCI spectrum illustrated in Fig. 1, with the exception of a small pen's: in the potassium window. This indicates approximately equal sodium and chlorine content, with pot tssium content consisting of less than 1% of the total electrolyte composition. A small peak in the calciu~ ~indow, not seen in lhis spectrum, is also often observed in perilymph spectra.
ferent from the spectra of endolymph in scala media. Both show very low levels of sodium, and equally high levels of potassium. Cr'/stal~ were not regularly encount,ered in the spaces of the organ of Cort ~. Crystals which were observed occurred at the edges of breaks in the reticular lamina which were produced during dissection. Since the EDXA spectra of such crystals were invariably
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2 3 4 5 key Fig. 3. Endolymph residue on the two sides of the rectorial membrane. Fluid residue associated with
the rectorial membrane, with corresponding EDXA spectra. The upper surface of the membrane (left) is marked by radially oriented ridges following freeze-drying. The EDXA spectra of the crystal~ scattered across this surface displayed equal emission in the chlorine and potassium windows, with very tittle emission in the ~dium window. ]'his indicates a relative excess of potassium over chloride, by
comparison with tl~e gC1 spec~rum of Fig. 1, and very low sodium content. The underside cJf the rectorial membrane (ri~t) shows ropey strands and frequent surface disconthzuities, as previously reported by Ross [19]. Hen~n's stripe is visible in the center of the figure. EDXA spectra of fluid crystals adhering to the underside of the membrane are identical to those of crystals on the uppe~ surface, indicatJxig that the fluid occupying the subtectorial space is endolymph. The crystals from which ~he illustrated spec~:a ~:ere obt',~ed are indicated by squares, which also repre~nt the counting a~ea. endoiymphatic, it is likely that those crystals seen in the organ of Cor~:i after dis~ctiori represent contamination from scala media. On the other hand, an amorphous; substance of zelatively low density was quite frequently observed bridNng the smaller spaces between ceils of the organ of Corti. Fig. 5 illustrates this substance in the spaces between
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Fig. 4. Otoconia and vestibular endolymph residue. Otoconia| crystals and endolymph residue isolated from the utricle, with associated EDXA spectra. The EDXA spectrum of the otoconium shows only calcium, since the carbon and oxygen of the ~rLonate radical emit X-rays which ~ n n o t be detected by EDXA. The er, dolymph spectrum indicates almost equal emission in the chlorine and pota~ium windows, with very little emission in the sodium window. A small sulfur peak is also visible. The spectrum is virtually identical to t h o ~ obtained t~om endolymph crystals in scala media, as illustrated in Fig. 3.
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Fig. 5. 'Cortilymph' residue. Secondary and backscattered electron images of the first row of outer hair cells. In the secondary image, note the amorphous substance which fills most of the gaps between adjacent cells. Stereocilia, when not lost during dissection, appear well preserved, in the backscattered image, the bolders of the outer hair cells appear intact beneath the quid residue. Outer hair cell nuciei are also visible in this image.
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Fig. 6. Preservation of cell membrane: in the organ of Corti. The first row of outer haft cells, with I)citer's cell cups still attached. Little t aid residue is visible in this region. The surface membranes of the outer hatr cells appear intact. Those ~f the Deiter's cells show numerous pits and sonic surface discoxltinuities.
a row of outer hair cells. The backscattered image shows the integrity of the cells beneath this substance. Cochlear tissues examined after rapid freezing and freeze.drying showed a level of structural preservation similar to that noted by other investigators [6,25]. At the resolving power of tile SEM, the limiting membranes of most cells showed relatively few discontinuities. This was especially true of cell borders which were adjacent to fluid spaces. Thus stria va:~cularis, Reissner's membrane, outer hair cells, pillar cells and tunnel crewing fibers showed essentially complete preservation of cell surfaces which fronted fluid. An exception to this rule were the cell bodies of Deiter's cells, which frequently showed discontinuities, as previously noted by Thalmann et al. [25]. Fig. 6 illustrates the preservati~m of cell ~embranes in the organ of Corti. The first row of outer hair cells is shown, wid~ the Deiter's cell cups s'till attached. Outer hair cell shape and cell membranes are well preserved. Fig. 7 ~lows a surface view of stria vascularis in secondary and backscattered SEM images. Fhe cells of the first layer are clearly outlined. Cell surfaces are continuous. Crystals of endc.lymphatic origin can be seen clearly in the backscattered image, and to a lesser extent in the secondary image.
|:ig. 7. Stria vascuhris. The scala media surface of the stfial epithelium is shown in secondary and backsc~-~1tered electron images. In the se,'ondary image, il~di~4dua| epithelia| ceUs are visible in relief, as are scattered crystals of endolymph residue. The surface membranes of the stria appear i n t ~ : : .... In the backscattered/~,~age, the membranes separathng adjacent cells -_re visible. Nuid residue is far more apparent in the backscattered/mage.
12
Fig. 8. Preservation of outer hair cells. Outer hair cells broken across their longitudinal axis during dissection. I.imiting membranes appear well pre,;erved, and the vacuoles in the hair cell cytovlasm are in the order of ! lanl or less. The surface membraaes of the adjacent pillar cells exhibit obvious breaks in the cell membranes. Tile i1~terit~r features ~f fleeze-dried cells could be observed with secondary SEM imaging, ~t breaks in tissue s:lmples which occurred during dissection. Large vacuoles are prescott in tile interior of soine cells, especially Deicer's cells and tile heads of pillar cells. P o f ti,ms t~f these cells closest to fluid surfaces tended to show smaller vacuolization. Fig. 8 sl~ows an interior view of outer hair cel~s brokea across their major axis during dissection. Vacuolization of outer hair cell cytoplasm is considerably less than that seen in Deiter's c~'lls or pillar cells, and the limiting membrane appears well preserved. The cot~dition of interior features of intact cells could be evaluated by backscattered
Fig. 9. Organ of Corti. Secondary and backscattered electron ~nages of the organ of Corti and the rectorial m e m b r a n e . Tne ~issue has spin a u r m g dissection just outside of the third row of outer hair ceils. In the secondary image, note the numerous, large discontinuities in the Deiter's cell m e m b r a n e s . O n l y a few sm;fl~ bre ~, ~ ,':~;,~ ~,e ,:ecn h~. outer h a l~ cell m e m b r a n e s . An a m o r p h o u s substance is present between some of the outer hair cells. In the backscattered image, the cell m e m b r a n e s beneath this s'abstance appear to be intact. Interior features of the tissue, such as cell nuclei and the borders between adjacent Deiter's calls, :.,re also visible. Dense crystals are scattered thickly across the surfaces of the tectorial m e m b r a n e and reticular lamina. A smaller n u m b e r of crystals c:m be seen on the surfaces of organ of Corti cells. Such crystals invariably exhibit the e l e c t r o b t e ratio of e n d o i y m p h . T h e h relationship to b~caks ia the reticular lamina suggests that the,, are displaced e n d o l y m p h crystals from scala media.
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Fig. 10. Reticular hmina. Backseattered electron image of the surface of the reticular lamina, The borders of tile reticular plates of the outer hair ce||s and the phalangeal processes of Deiter's ee~ are visible in a manner suggestive of a light-microscopic image of a surfa~ preparation. Fluid residue crystals are scattered across the lamina. The EDXA spectra of these crystals are invariably characteristic of endolymph. The reticular lamina is broken near the lower edge of the figure, revealing the crystal-free surI~ces of the outer hair cell bodies.
SE~'i imaging. Fig. 9 shows flae organ of Corti in both secondary and baekscattered images. The backscattered image reveals mm~y details which cannot be seen in ~ e secondary image. The borders between adjacent Deiter's ceils are clearly demarcated, and the nuclei of both outer hair cells and Deiter's cells are viable. Cryst~s preei#tated from coch|ee- fluids are densely scattered across the reticular lamina and tectorial membrane. Backscattered imaging of interior cell junctions was of great value in orienting on the
15
surface of the reticular lamina. When the cell bodies of the outer hair cells could not be visualized, their position within the organ of Corti could not always be ascertained from e~ation of the surface of the reticular lamina, due to the fluid residue adhering to the surface. Backscattered imaging of the lamina revealed the pattern of cell junctions, and resembled the image ob~rved in a surface preparation of the organ under light microscopy, as shown in Fig. 10. DISCUSSION
Five major conclusions may be draw from the data presented in this report. (1) Our ~atomical observations suggest that the integrity of the major extracellular compartments of the inn~.~rear is maintained during freezing and freeze-drying. Moreover, our EDXA data indicate that high-purity samples of fluid residues may be obtained rou. tinely by microdissection of freeze-dried cochleas. (2) The fluid in the sabtectorial space appears to be identical to endolymph. High levels of sodium, previously reported at this location [ 19], were net observed. (3) Endolymph obtained from the utricle exhibits the same low sodium content observed in the scala media. Previous reports of differences in the sodium content of vestibular and cochlear endolymph [ 18,21] may reflect the difficulty of obtaining uncontam. inated samples. (4) After freeze-drying, the fluid which fills the extracellular spaces of the organ of Corti leaves a residue which is distinctly different from those of either perilymph or endolymph. The anatomical appearance of this residue is consistent with a significantly higher protein content in the fluid filling the organ of Corti spaces, as suggested by Engstrom
[6]. (5) Backscattered SEM imaging was found to be an invaluable tool for th~~,evaluation of interior features of tissue samples and for the. detection of crystalline fluid residues. Freeze-drying is commonly employed to immobilize ions for :he later collection of samples from complex biological tissues [ 17]. This technique has the obvious advantage of eliminating ion movement due to fluid displacement or diffusion, greatly r,ducing opportunities for sample contamination. While in rive electrolyte-volume relationships of fluids are lost during freeze-drying~ the rdative proportions of electrolytes ca.a readily be measured by conventional elemental analysis teelmiques or by EDXA. The cochlea, almost completely enclosed in bone, is not an obvious c~didate for cryogenic freezing. It is perhaps for this reason that few studies have attempted to utilize freezeMrying to iramobilize ions in the cochlea. The difficulties pre~ented by the freeze-dried preparation of samples are many. Perhaps the most significant potential problem is the formation of large ice crystals during freezing. To minimize the size of ice crystals, investigators have used a variety of cryogenic fluids. Although extremely cold (-196°C), the heat transfer characteristics of liquid nitrogen make it an unsuitable heat sink. Organic compounds like Freon 12 or isopentane cooled ha liquid nitrogen have e ~ g rates o f - l ~ - I ~ ° C / s and have been used by many investigators [ 17]. Regardless of the c o o ~ g rate of the immersion bath, movement of heat out of a bioIogic~ specimen is limited by the relatively poor conduction characteristics of water, the primary biological so|ve~t. Thus it is difficult to ac~eve rapid
16 freezing in large biological ~ p l e s . In the ~ e a , the bony ~ ~ fu ILn~its heat " o transfer. However, s u b d i ~ o n of the coctflea or even t ~ ~ of f ,zb~e~ ~ ° would produce greater damage to ion cornpmmen ts than freeze artifact. ~ orgy ~ . able means ~f freezing the cochlea is to quench Me inner e2r w~hole, et at. [25] have estimated that freezing of the intact c.ochlea in Freon 12 cocked to -159°C is not complete until about 1.5 s after quenching. This freezing time undoubtedly a~ow~ ice G crystal formation in cochlear fluids and tissues. The uz~ o f ~ e 1¢ecrystals formed, ~ d the amount of physical damage c a r d , would d e ~ d upon the e ~ t f r e e ~ g time -topoint within the cochlea as weU as the protein and lipid ~ n t of the p ~ ~ fluid or tissue in question. The signifiem-lt questions for the use of freezing and subse t freezedrying in the analys/s of ion content of ¢ochle~~fluids and c , ~ is ~ degree to which ice crystal formation causes the m o v e ~ n t of ions out of the p~tions ~ they ~ g ~ y in rive. An ~spcct of ice crystal formation wMch could potentially alter ion c o m ~ t m e n t boundaries is the breakdown of ceil membr~e~ ~ d associated cetl-cgH junctions during " n , and then during intra~llular crys~l formation. ex,racellular crystal formatlo Fluid and tissue cooling, and thus ice ¢ryst~ formation, pt~.'~mab|y ~ s with ¢och. lear location. The most rapid cooling probably occurs close to ,he codflear bone, ~ ,pirM ligameni and stria vasoularis, and in perilymph. Cooling of more cgntr~ly located struc. tures such ~s the organ of Corti may be significantly slower. Oar anatomie.al ob~r~ltions suggest that heat loss may occur more rapidly in tissue surfaces adjacent to fl~d, ~ c e vesiculation of cytoplasm is smallest at this location. The slowest cooling ap~ars to occur at the center of large, contiguous cell groups like the Dieter's cells. Freezing of t ~ from fluid boundaries inward may limit the degree to which ice cryste~ls penetrate cell memb,anes, d'ae to smaller crystal size and possibly orientation of cry~al pl~me, parallel to men~brane surfaces. An important consequence of the apparent p:e~rvation of cochlear ~el[ membranes adjacent to fluid is the potential that fluid compartment integrity is maintained during freezing and freeze-drying. It seems likely that both intrac~ltular and extracellular ion distribution would be maintained if there were no major defects in the cell membrane. Even with small membrane discontinuities, it i.~ possible that movement of ions is limited to a small region around the membrane defect, ~ c e both tmu¢ and fluid would be in the process of freezing at the time of membrane penetration. ~n addition to ice crystal formation, another potential source of ion movement is the freeze-drying process itself. Even in a tissue matrix, in w~dch ions are probably bound to various protoplasmic constituents, only aepression of temperature to the eutectic point of water ( - 130°C) can absolutely suppress ion movement. Sublim~tio~ of water has been observed to cause slight movement of ions in tissue even when temperatures are main. rained below -80°C [4]. However, given the size and the bony capsule of the intact cochlea, the use of extremely low temperatures in freeze.drying is :~:,t practical. Whe~ water ~s removed at the moderatly low temperatures (-40°C) necessary for cochlear free~dryh~g, some ion movement is to be expected, especially in the case of fluid with a reintively low protein and lipid conlent such as endolymph or periiymph. |t is not surprish~g, therefore, that the contents of the cochlear scalae after freeze.drying are not uniform, ~hhfly.scattered ions. The degree to which ions condense into solid masses during freeze. drying reflects the size and configuration of proteins mad lipids w~ch migh~ form a matrix for the dehydrating electrolytes.
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that ion movement due to cell membrane puncture
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repeated obsemtions on isolated fluid residues from the codflea, we have fuqat spectra of endolymph residue indicate high levels of potassium, somewhat lower levels of chlorine, and very low leveis of sodium. EDXA ,,~pectra obtained from perflymph residue show approximately equal levels of sodium and c~orine, with trace of ~dealcium,/The electrolyte content of the two major cochlear ~in by EDXA spectra are consistent with the best values which have been by other assays. ~ e extols of potassium over chlorine in endolymph has been in ~¢veral mammMian species [7,14 ~23,24]. The equal levels of sodium and c h l o ~ e which w~~.observed in perilym?h of the chindlla are similar to those seen in the eat [7,14,23] and ~ e the relative excess of sodium observed in the guinea pig [7,24]. The low levels of sodium in endol~zmph spectra and of potassium in perilymph spectra indicate no s ~ f i c a n t admixture of the fluids filling the three cochlear scalae during freezing or freeze.drying. Furthermore, mierodissection of freeze-dried inner ears has routinely provided samples of endolymph a~d perilymph with a high order of purity. This is in contrast to many ~tudies based upon invasion of the cochlear fluid spaces in a hydrated state [2,12-14,17]. We conclude chat the limitations of our methodology, such lo~ of absolute electrolyte-volume rela,ionships in fluid residues, are outweighed by the low levels of sample contamination which can routinely be achieved. EDXA spectra obtained from fluid residues at various cochlear locations have implications c o n e e ~ g the distribution and phyziology of endolymph. Our data provide strong evidence that the fluid which occupies the space between the rectorial membrane and the reticular lamina is endolymph. Flock [6] ri~easured chlorine and potassium levels in the subtectorial ~ace utilizing EDXA. He observed significant levels of potassium, and inter. preted this to reflect the presence of endd:Jmph. He did not, however, measure sodium levels. Ross [19] obtained EDXA spectra fr3m both sides of the rectorial membrane. She reported significant levels of sodium m s~ctra obtained from the underside of the membrane, in addition to potassium, and concluded tttat a fluid similar to perilymph must occupy the subteetorial space. In direct conflict with the data of Ross [ 19], our spectra. show equally high levels of potassium, aLd low levels of sodium, in fluid residues on both ~des of the rectorial membrane. In no inst~ce have we observed significant levels of sodi. um emi~on in spectra obtained from the ~eetorial membrane or from fluid residues associated with the membrane. EDXA ~ c t r a obtained from cochleal and utricular endolymph show equally Mgh pot a ~ u m and low ~dium content. It has beer~ suggested by several authors that the electrolyre compoation of ~ndolymph is different in the two divisions of the inner ear, based upon assays of fluid samples obtained by mieropipett¢ penetratiom of the endolymphatic compartment [18,21|. Separate origins and circulations of cochlear and vestibular endolymph were also p r o I ~ d . Our data do not support a difference between endolymph in the two divisions of the labyrinth. Differences reported in previous investigations may represent the difficulty of obtaining uncont~rlinated samples of endolymph from the sac"_fhe residues of Lrmer ear fluids were distinct in the~ anator~ica] appearance, presumably becau~ of differen~s m their protein and ~pid content, as well as *_hesize and
18
distribution of ice c r y ~ produced during freez~g. P e v 3 ~ ~ n t P y e ~ b i t e d the greatest degree of condensation durizlg f r e e z e ~ i n g , frequently ~ in scab tymp~i and vestibuli empty of fluid residue. ~ ~ e s t s a rels low pr ~ m or lipid content. Endolymph, in c o n t r ~ , ~.ally precipitatedas an ~ributed, cottony mass which completely ~ the sca~ media. ~ ~ a higher protein or lipid content than perilymph. Comparison of EDXA spectra obtained from the two fluids shows that while endolymph spectra c ~ t e n t l y show m e a s u m b ~ ~ o n sulfur window, suggesting protein, con~derably ~ ~ o n in ~ w i n ~ ~ observed in purllymph spectra. This observation is not c o ~ t with most previous e~imates of the prorein content of endolymph and pe~ymph, in w ~ ~ latter has been ~own to exhibt a higher protein content [7,16~23]. However, these observations were ~ performed with micropipette sampling. Tonndorf et el. [26] and Rauch [15] have p r e s e n ~ anatomical evidence that a matrix of glycoprotein is present in scala media. Such a matrix might not be sampled during withdrawal of fluid through a small aperture pipette, but would certainly be included in our micro-dissected ~mples. Our data may reflect the presence of s, ch a glycoprotein in scala media. Both pertlymph and endolymph displayed characteristic crystals ~s part of their fluid residues, The appearance of cryst~s as refidues of freeze-drying has no~ aiwsys been reported from the cochlea. Flock [6] described the fluid residues as cottony muses of filaments or plates. Th~ma~n et al. [25] mentioned fluid residues but did not describe them. However, Peterson et al. [ 14] observed crystals m freeze.dried droplets of undolymph and perilymph which had previously been withdrawn from the inner ear in micropipettes. It seems possible that the failure of some investigators to report crystals in cochlear fluid residues resulted from the lack of ability to detect high-denfity particle.s embedded in the residue matrix. We have found backscattered imaging to be the only reliable means of identifying crystal presence ~ fluid residues or on tissue surfaces. The ~ack of crystalline fluid residues in ~ e organ of Corti ~paces indicates that the fluid which normally occupies the spaces may have a higher protein or lipid content than either perilymph or endolymph. It seems unlikely that the restricted size of the fluid spaces in the organ would restrict crystal formation, since cryst~ were ~way$ abundan t in the subtectorial space and inner sulcus. Engstrom [5] has suggested that the protein content of 'cortilymph' maybe higher than that of the other cochlear fluids, on anatomical grounds. High protein content is also characteristic of e x t ~ H u l a r spaces with restricted fluid circulation. This might well apply to the spaces of the organ of Corti. The data obtained in the present investigation leave many important questions concerning cochlear ion distribution, and the effects of freeze.drying upon that distribution, unanswered. While it seems clear that there is no gross movement of ions between the central regions of the cochlear scalae, we have not determined the degree to which ions may move on a smaller scale. This would include ion movement from one cell to another, as well as from the interiors of cells ~o the edges of fluid compartments. T1t$ extent of such movement is of particular importance to the measurement of ion distribution in the fluid spaces of the organ of Corti, as wall as in cochiear t~ues. The variability which we have observed m EDXA measurements of element content has been quite low. This suggests that more quantitative use of E D X A is feasible in the inner ear, providing that appropriate controls are employed. We are currently exploring
19
the rive us~ of EDXA, to characterize ch~ges in cochlear ion distribution under raeh conditions as noise exposure, drug treatment and anoxia. REFERENCES [1] Bosher, S.~-. and Warren, R.L. (1968): Observations on th~ electrochemistry of the cochlear endolymph of th, rat. Proc. R. Soc. London 171,227-247. [2] Citron, L., Exley, D. and Hallpike, C.S. (1956): Formation, circulation and chemical properties of labyrintl2ine fluids. Br. Med. Bull. 12, 101-106. [3] Citron, L. and Exley, D. (1957): Recent work on biocllemistry of the labyrinthine fluids. Proc. R. Soc. Med. 50, 697-701. [4| Dorge, A., Rick, A., Gehfing, K., Mason, J. and Thurau, K. (1975): Preparation and applicability of freeze-dried sections in the microprobe analysis of biological soft tissue. J. Microsc. 22, 205214. [5] Engstrom, H. (1960): Corfilymph. The third lymph of the inner ear. Acta Morphol. Neerl. Scand. 3, 192-204. [6] Hock, A. (1977): Electron probe determination of relative ioI~ distribution in the inner ear. Acta OtolaryngoL 83, 239-244. [7] Johnstone, C.F., Schmidt, R.S. and Johnstone, B.M. (1963): godium and potassium in vertebral cochlear endolymph as determined by flame microspectropho :ometry. Comp. Bioehem. Physiol. 9, 335-358. [8] 3uhn, S.K. (1971): Perilymph findings in experimentally indue :d acidosis. Arch. Otolaryngol. 93, 384-387. [9] Lawrence, M. (1967): Elec~:~cpolarization of the tectorial metnbrane. Ann. Otol. Rhinol. LaryngoL 76, 287-312. [10] Lawrence, M., Nuttal, A. aad Clapper, M. (1975): Electric~l potentials and fluid boundaries within the organ of Corti. J. Acoust. Son. Am. 55,122-1~8. [I1] Lira, D.J. (1972): Fine morphology of the teetorial membrare; its relationship to the organ of Corti. Arch. Otolaryngol. 96, 199-215. [12] Miyake (1960): Biochemical study of labyrinthine fluids. J Otorhinolaryngol. Soc. 3ap. 63, 1-18. [13] Oliveim, C.A., Juhn, S.K. arid Kim, C.S. (1077): The effect of microembolization of cochlear capillaries on the ionic composition of perilymph and endolymph. Trans. Am. Aead. Ophthalmol. OtolaryngoL 84, ORL 9C8. [14] Peterson, S.K., Frishkopf, L.S., Lechene, C., Oman, C.M. and Weiss, T.F. (1978): Element corn-" position of inn~ ear lymphs in cats, lizards, and skates determined by electron probe microanalysis of liquid samples. J. Comp. Physiol. 126, 1-14. [15] Ranch, S. Unpublialed observation, cited in Tonndorf et al., op. tit. [16] Rauch, S. (1964): Biochemie des Hororgans: Einfiihrung in Methoden und Engebnisse. G. Thieme Verlag, Stuttgart. [ 17] Robards, A.W. (1974): Ultra~tructural methods for looking at ceils. Sci Prog. Oxford 61, 1-80. [18] Rodgets, K. and thou, LT.Y. (1966): Concentrations of inorganic icns in guinea pig inner ear fluids. 1: Concentration of potassium and sodium in cochlear and utricvlar endolymph. J. LaryngoL 80, 778-790. [ 19] Ross, M.D. (i974): The rectorial membrane of the rat. The Am. J. Aria ~.. 139,449-4:32. [20] Schuknecht, H.F. and McNoiU, R.A. (1966): Light microscopic observ ltions on the pathology of endolymph. 3. LaryngoL 80, t - 1 0 . [21] Setlick, P. and 3ohnstone, B. (1975): Production and role of inner ea::~fluid. Prog. Neurobio]. 5, 1-18. [22] Seymour, 3.G. (1954): Ob,ervations on the circulation of the cochlea. 5. LaryngoI. 68,689-697. [23] Silverstein, H. and Griffin, W.L. (1970): Compaxi~n of inner ear flui is in the antemortem and po~mortem state of the cat. Ann. Otol. Rhinol. Laryngol. 79, 178-t86.
20 [24 | Smith, C ~ . , L ~ O~H. ~ W La~Tngo~cope ~ , ~ A I - I ~ [25] Th~llman, Ro, Comeg'/s, T.H. and fr~z~-dri~ time for u[ttan~ictoch [26] Tonndoff, J., DuvaH, A..I. and Re~ various vita~ ~tain~. Ann. OtoL RhinoL ~ ~ o ~ .
71,801-84I.
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elect~on im were c~mbined with energy d~spcrsi,~e ribuflon in z,Mried~ir~ch~ inner ears. Fluid residues ~ of mOteristic~lly shaped crys-::~ ~~d from Crys~ls ~m consistem-withthe ~ .In , um lov©lsin perflymph and sodi: ~&at~ ty of the major fluid comp~.rtments
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,~ e~n~y inenti,-~ ,o those ob~inocl from ¢ochle~ :in ionic compo~flon of e~dolymph in the two divisions of the of the organ of Corti does not form crystals, w~ch suggests that the ~ ph or endolymph. Secondly scanning electron n of c~L~membrancsof most cochtea~ tissues, features of t'~ue0 such as interior cell ~tered scanning electron ima~dng was ' the identification of crystai~ of fluid
two major fluid ad ~v¢~al miall~r spaces nments. In addition, the ays ~ impoxtant role in ,
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that endolymph ir~s variety of species contains approximately 150 mEquiv./l of potassiu m and I l0 mEquiv./l of chlorine. However, there has been fa~ less agreement concerning t~he level of scdiura in this fluid, with values ranging from 1.0 to 1i5 mEquiv./l [1,7,12, 14,!8,24]. It seems likely that the lowest measurengmts of closer to t:le ~ctual level. Higher levels presumably reflect the uncontan~n~ted sample of endolymph with the invasive techniques currently employed. Both perfiymph and endoIymph contain about 6 mEq~v.]l of calcium, and traces of other inorg~c ions [3,14,18]. Protein concent~tion of the two fluids has ~ measured at 1-2% of the level in senan, with perilymph c o n ~ more protein than ~ d o lymph [2,16,23]. However, vital staining of the cochlea has ~oduced e v i ~ that a glycoprotein ~latfix may be suspended wihhm the endolymphafic ~ace [2,5]. It has been su~ested on s~atomical grounds that endolymph in the utricle and cochlea may circulate in.dependently [20-22]. In support of this b y , thesis, Rodgers and Chou [18] reported that sodiv.m levels in scala media are higher than those in the utride. Conversely, Sellick a~.d John~tone [21 ] concluded in a renew of several st~Jdiesthat sodium levels are higher in vestibular eadolymph than in cochlear endotymph. The ionic composition of the fluids filling |he various subcompartments of the cochlea - such as the inner sulcus, the subtectoriat ~pace and the extracellular spaces of the organ of Corti - have not been established with certainty. Anatomi~ data obtatined by Engstrom ~5] led him to suggest that organ of Corti fluid spaces are filled with a fired with an electro|y~:e composition similar to perilymph, but with a significantly ~ r prorein concentr~fion. He termed this fluid 'cortflymph'. The fluid filling the subtectorial space and the inner sulcus has been variously identified as endolymphati¢ or peri. lymphatic in composition, based on anatomical [I 1,19], physiological [9,10] and X-ray microanalytic [6,19] grounds. The intraceUular ionic composition of hair cells and stria vascularls cells have not been directly assessed. However, given our current understanding of intrscellul~ environments, it seems likely that potassium, chlorine and organic cations are the dominant electrolytes. The major difficulty inherent in the measurement of the ionic composition of fluids in small extracel~ular compartments, and even in some relatively large extraceHular compa~ments, is fluid contamination from one compartment to another during ~ p l i n g . The wide range of values of sodium content measured in samples of endolymph [ 1,2,7,12-~14, 18,23,24] attests to the seriousness of tt'ds problem. Since fluid sample collection necessi. tares the violation of fluid space integrity, techniques have concentrated upon minimizing opportunities for ion movement. The use of glass micropipettes has been the ~ d ~ d mode of obtai:ning fluid samples, but the success of this method is quite v~able. $in~at objections can be made concerning ion.sensitive microelectrodes. Oliviera et al. [13] employed cryogenic freezing of the cochlea and dissection of fluid samples in a frozen. hydrated state. However, sodium levels obsorved in endolymph with this technique were about 40 mEquiv./l, again suggesting contamination. Cryogenic fr~ezing followed by freeze-drying is commonly employed to immobilize " 'o n of freeze-dried"tautenS" l ~ the ohions in complex biologic specimens [17]. Di~ct~: vious advantage that sampling is performed in an anhydrous state, e l ~ a f i n g ion movement due to fluid displacement or to diffusiono However, the bony capsule of ~he e ~ e a necessitates freezing of the inner e~ intact. Whether such freezing in fact preserves the
integrity of e~hlear fluid compartments is not clear, especially given the data of Oliviera et el. [13]. The size of the intact cochlea necessitates freeze-drying at moderately low temperatures (--40 o C). !t has been demonstrated that such temperatures allow some movement of ions during the freeze-drying process [4]. Whether movement of ions during not known. Wtfil~ some freeze
METHODS
Normal chinchillas (Chinchilla laniger) were mesthetized with sodium pentobarbitol (?0 mg/kg) and ketamine an~ the cochleas rapidly dis~cted from the each subject:- was taken within 60-90 s, and second within 120-180 s, of decapitation. Care was taken not to breach the round and oval windows during dissection. The whole cochlea.s were then quenched in Freon 12 cooled to its liquid-solid interface (-159°C) ~ d freeze-dried at 0.01 Torr and -40"C for at least 72 h. After freeze.dzying, specimens were maintained under vacuum over calcium sulfate. All samples were di~cted with instruments which had been washed with #ass-distilled water followed by anhydrous acetone, Specimens for ~ e present inv~stigation were obtained from the upper basal and lower second cochlear turns of the cochlea, ,rod the utricle. The bony capsule of tbe inner ear was fractured, beginning at the medial margin of the round window, opposite the ovfl window. The bone was then pid,:ed away with fine scalae with forceps and probes. ,,ments. The free juaction of o,g ofco,ti ofte be further the outer ~lls ~,outer hai~: d ~ c t e d by ~p~ati ~gments ~ticells w i ~ or wi~ou l ~ g the methods ( ~ spiral i:iga. ment be di~eted as a U~t, or stria could hi; ~p~atea tram the un~eriying ligament by ~ r t i n g a razor-blade fr and twistir g, which caused 1:he two tissues to separate naturally as de~ribed by Tlalmarm et el. [25 ]. Outer h ~ cells from the second turns could not be isolated under light mi :roseopic v i s u ~ t i o n . However, when lon~tudinal fragments of the ~rgan of Corti were identified ruder ~ing electron micro~ope as having only outer hmr cells, them fra~ents could later be removed from the SEM stub md tea~d apart to obtain mdivid-
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Fig. I. EDXA spectra of N ~ l and KCI controls° EDXA spectra obtained from crystals of t~ze-dried physiologic salt solutions, a. Reagent-f,rade NaC|. The spectrum reflects the differential emission of X-rays from sodium as compared to chtorine, Approx~ately four times as many emissions are observed in the chlorine window as are seen in the sodium window, when the two eleme~its a ~ pre~nt in equal proportions, b. Re~ent-grade KCI. Cldorine emits about 20% more X-rays than potassium, when the levels of the two elements in the excited c r y s ~ are the same,
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All specimens were mounted on carbon planchets with plastic cenent. They were then coated with carbon. Carbon coating gives secondary scanning images similar to those seen with heavy metal coating, but does not interfere with backscattered imaging or EDXA as does the metal coating. Imaging of specimens was performed an an E I'EC Auto~can SEM. Instrument parameters included an accelerating voltage of 20 keV, ,::ondensor current of 2.5 A and working distance of' 16 mm for secondary ima~ng, and a condenser current of 1.8 A and working distance of 13 mm for backscattered imaging. 'i~le ETEC Autoscan was equipped with a KEVEX Quantex system for EDXA. A conden~or current of 2.0 A, working distance of 13 rnm and tilt of 45 °, was employed for all EDXA measurements. Specimen current varied from 0.25 to 0.75 hA. Dead times were liraited to between 30 and 70%. Initial work demonstrated that we routinely could exp~;ct K~ peaks in the 1.01-1.09 keV (Na), 1.98-2.06 keV (P), 2.27-2.35 keV (S), 2.59-2.67 (CI), 3.283.36 keV (K) and 3.66-3.74 keV (Ca) windows. Both a matrix corre~-tion factor and correction against an adjacent r e , o n of carbon and glue were used to e iminate background emission initially, but the measured background was ~,~eleetedfor routine use. EDXA dala were coflected at a magnification of 15 • 10a from 0.5-2.0 tam crystals of perilymphatic or endolymphatic origin. The emission of X-rays, measured as counts/s or as peak.to-backgz ound ratio, does not provide an absolute measure of element abundance. The atomic nu~ber of an individual element, interactive effects between different element:~, specimen de~sity and other operating factors can influence tke number of emitted X-rays which are d.te~ted from a given specimen. To provide a simple standard for comparison with cochlear fluid residues, droplets of 0.9% solution of reagent-grade NaCI and KCI were frozen ant freez'-dried on carbon stubs, and prepared for EDXA in the same manner as cocMea~ specimens. Typical EDXA spectra obtained from the resultant crystals are shown in Fig. 1. These spectra illustrate the differential emission of X-rays from the different eleme~ats which are of partitular importance as electrolytes in the inner ear. '
,~
,,~
RESULTS
The fluid resid~.les which remained after freeze-drying differed with their location. Crystals in a tenuous matrix were found in isolated clumps within the perilymvaatic spaces of scala tympani and vestibuli. The crystals varied widely in size and were roughly cuboidal in shape. Fig. 2 illustrates isolated perilymph residue, with an associated EDXA spectrum. The spectrum indicates approximately equal peak-to.background levels of sodium and chlorine, with small but measurable quantities of potassium and calcium. Fluid residue in the scala media frequeltly formed a cottony mass which filled the scala. Under SEM, this residue was similar in appearance to the perilymph residue, except that the crystals tended to be slightly elongated. Fig. 3 shows fluid residue on the upper and lower surfaces of the rectorial membrane, with associated EDXA spectra. Both spectra display a relative excess of potassium e~er chlorine, and an extremely low level of sodium.
Fig. 4 ~ustrates isolated fluid residue and otocon~a from the utficle. EDXA spectra obtained from an otoconial crystal and an endolymph crystal are also shown. As illustrated in the figure, endolymph spectra obtained from the utricle are not noticeably dii-
80 U
40 0
0 0
I
2
3 keV
4
5
Fig. 2. Freeze-dried perilymph residue. Secondary and backscattered electron images el clumped perilymph crystals, isolated from u:ala Wmpani of the freeze-dried inner ear. The crystals are bour~d together by small amounts of an ~tmorphous matrix. The EDXA spectrum was obtained from the small crystal h~dicated by the arrow, the larger masses being inappropriate for EDXA due to suppressed emission of X-rays from sodium. The inner border of the box surrounding the crystal in the seco~,~dary electron image represents the region excited hy the electron beam during EDXA. The perllymph '~pectrum i:~ essentially identical to the NaCI spectrum illustrated in Fig. 1, with the exception of a small pen's: in the potassium window. This indicates approximately equal sodium and chlorine content, with pot tssium content consisting of less than 1% of the total electrolyte composition. A small peak in the calciu~ ~indow, not seen in lhis spectrum, is also often observed in perilymph spectra.
ferent from the spectra of endolymph in scala media. Both show very low levels of sodium, and equally high levels of potassium. Cr'/stal~ were not regularly encount,ered in the spaces of the organ of Cort ~. Crystals which were observed occurred at the edges of breaks in the reticular lamina which were produced during dissection. Since the EDXA spectra of such crystals were invariably
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u
2 3 4 5 key Fig. 3. Endolymph residue on the two sides of the rectorial membrane. Fluid residue associated with
the rectorial membrane, with corresponding EDXA spectra. The upper surface of the membrane (left) is marked by radially oriented ridges following freeze-drying. The EDXA spectra of the crystal~ scattered across this surface displayed equal emission in the chlorine and potassium windows, with very tittle emission in the ~dium window. ]'his indicates a relative excess of potassium over chloride, by
comparison with tl~e gC1 spec~rum of Fig. 1, and very low sodium content. The underside cJf the rectorial membrane (ri~t) shows ropey strands and frequent surface disconthzuities, as previously reported by Ross [19]. Hen~n's stripe is visible in the center of the figure. EDXA spectra of fluid crystals adhering to the underside of the membrane are identical to those of crystals on the uppe~ surface, indicatJxig that the fluid occupying the subtectorial space is endolymph. The crystals from which ~he illustrated spec~:a ~:ere obt',~ed are indicated by squares, which also repre~nt the counting a~ea. endoiymphatic, it is likely that those crystals seen in the organ of Cor~:i after dis~ctiori represent contamination from scala media. On the other hand, an amorphous; substance of zelatively low density was quite frequently observed bridNng the smaller spaces between ceils of the organ of Corti. Fig. 5 illustrates this substance in the spaces between
20
40"
0
I
2
keV
3
4
5
Fig. 4. Otoconia and vestibular endolymph residue. Otoconia| crystals and endolymph residue isolated from the utricle, with associated EDXA spectra. The EDXA spectrum of the otoconium shows only calcium, since the carbon and oxygen of the ~rLonate radical emit X-rays which ~ n n o t be detected by EDXA. The er, dolymph spectrum indicates almost equal emission in the chlorine and pota~ium windows, with very little emission in the sodium window. A small sulfur peak is also visible. The spectrum is virtually identical to t h o ~ obtained t~om endolymph crystals in scala media, as illustrated in Fig. 3.
0
C
Og
50"
IO0 o
Fig. 5. 'Cortilymph' residue. Secondary and backscattered electron images of the first row of outer hair cells. In the secondary image, note the amorphous substance which fills most of the gaps between adjacent cells. Stereocilia, when not lost during dissection, appear well preserved, in the backscattered image, the bolders of the outer hair cells appear intact beneath the quid residue. Outer hair cell nuciei are also visible in this image.
10
Fig. 6. Preservation of cell membrane: in the organ of Corti. The first row of outer haft cells, with I)citer's cell cups still attached. Little t aid residue is visible in this region. The surface membranes of the outer hatr cells appear intact. Those ~f the Deiter's cells show numerous pits and sonic surface discoxltinuities.
a row of outer hair cells. The backscattered image shows the integrity of the cells beneath this substance. Cochlear tissues examined after rapid freezing and freeze.drying showed a level of structural preservation similar to that noted by other investigators [6,25]. At the resolving power of tile SEM, the limiting membranes of most cells showed relatively few discontinuities. This was especially true of cell borders which were adjacent to fluid spaces. Thus stria va:~cularis, Reissner's membrane, outer hair cells, pillar cells and tunnel crewing fibers showed essentially complete preservation of cell surfaces which fronted fluid. An exception to this rule were the cell bodies of Deiter's cells, which frequently showed discontinuities, as previously noted by Thalmann et al. [25]. Fig. 6 illustrates the preservati~m of cell ~embranes in the organ of Corti. The first row of outer hair cells is shown, wid~ the Deiter's cell cups s'till attached. Outer hair cell shape and cell membranes are well preserved. Fig. 7 ~lows a surface view of stria vascularis in secondary and backscattered SEM images. Fhe cells of the first layer are clearly outlined. Cell surfaces are continuous. Crystals of endc.lymphatic origin can be seen clearly in the backscattered image, and to a lesser extent in the secondary image.
|:ig. 7. Stria vascuhris. The scala media surface of the stfial epithelium is shown in secondary and backsc~-~1tered electron images. In the se,'ondary image, il~di~4dua| epithelia| ceUs are visible in relief, as are scattered crystals of endolymph residue. The surface membranes of the stria appear i n t ~ : : .... In the backscattered/~,~age, the membranes separathng adjacent cells -_re visible. Nuid residue is far more apparent in the backscattered/mage.
12
Fig. 8. Preservation of outer hair cells. Outer hair cells broken across their longitudinal axis during dissection. I.imiting membranes appear well pre,;erved, and the vacuoles in the hair cell cytovlasm are in the order of ! lanl or less. The surface membraaes of the adjacent pillar cells exhibit obvious breaks in the cell membranes. Tile i1~terit~r features ~f fleeze-dried cells could be observed with secondary SEM imaging, ~t breaks in tissue s:lmples which occurred during dissection. Large vacuoles are prescott in tile interior of soine cells, especially Deicer's cells and tile heads of pillar cells. P o f ti,ms t~f these cells closest to fluid surfaces tended to show smaller vacuolization. Fig. 8 sl~ows an interior view of outer hair cel~s brokea across their major axis during dissection. Vacuolization of outer hair cell cytoplasm is considerably less than that seen in Deiter's c~'lls or pillar cells, and the limiting membrane appears well preserved. The cot~dition of interior features of intact cells could be evaluated by backscattered
Fig. 9. Organ of Corti. Secondary and backscattered electron ~nages of the organ of Corti and the rectorial m e m b r a n e . Tne ~issue has spin a u r m g dissection just outside of the third row of outer hair ceils. In the secondary image, note the numerous, large discontinuities in the Deiter's cell m e m b r a n e s . O n l y a few sm;fl~ bre ~, ~ ,':~;,~ ~,e ,:ecn h~. outer h a l~ cell m e m b r a n e s . An a m o r p h o u s substance is present between some of the outer hair cells. In the backscattered image, the cell m e m b r a n e s beneath this s'abstance appear to be intact. Interior features of the tissue, such as cell nuclei and the borders between adjacent Deiter's calls, :.,re also visible. Dense crystals are scattered thickly across the surfaces of the tectorial m e m b r a n e and reticular lamina. A smaller n u m b e r of crystals c:m be seen on the surfaces of organ of Corti cells. Such crystals invariably exhibit the e l e c t r o b t e ratio of e n d o i y m p h . T h e h relationship to b~caks ia the reticular lamina suggests that the,, are displaced e n d o l y m p h crystals from scala media.
14
Fig. 10. Reticular hmina. Backseattered electron image of the surface of the reticular lamina, The borders of tile reticular plates of the outer hair ce||s and the phalangeal processes of Deiter's ee~ are visible in a manner suggestive of a light-microscopic image of a surfa~ preparation. Fluid residue crystals are scattered across the lamina. The EDXA spectra of these crystals are invariably characteristic of endolymph. The reticular lamina is broken near the lower edge of the figure, revealing the crystal-free surI~ces of the outer hair cell bodies.
SE~'i imaging. Fig. 9 shows flae organ of Corti in both secondary and baekscattered images. The backscattered image reveals mm~y details which cannot be seen in ~ e secondary image. The borders between adjacent Deiter's ceils are clearly demarcated, and the nuclei of both outer hair cells and Deiter's cells are viable. Cryst~s preei#tated from coch|ee- fluids are densely scattered across the reticular lamina and tectorial membrane. Backscattered imaging of interior cell junctions was of great value in orienting on the
15
surface of the reticular lamina. When the cell bodies of the outer hair cells could not be visualized, their position within the organ of Corti could not always be ascertained from e~ation of the surface of the reticular lamina, due to the fluid residue adhering to the surface. Backscattered imaging of the lamina revealed the pattern of cell junctions, and resembled the image ob~rved in a surface preparation of the organ under light microscopy, as shown in Fig. 10. DISCUSSION
Five major conclusions may be draw from the data presented in this report. (1) Our ~atomical observations suggest that the integrity of the major extracellular compartments of the inn~.~rear is maintained during freezing and freeze-drying. Moreover, our EDXA data indicate that high-purity samples of fluid residues may be obtained rou. tinely by microdissection of freeze-dried cochleas. (2) The fluid in the sabtectorial space appears to be identical to endolymph. High levels of sodium, previously reported at this location [ 19], were net observed. (3) Endolymph obtained from the utricle exhibits the same low sodium content observed in the scala media. Previous reports of differences in the sodium content of vestibular and cochlear endolymph [ 18,21] may reflect the difficulty of obtaining uncontam. inated samples. (4) After freeze-drying, the fluid which fills the extracellular spaces of the organ of Corti leaves a residue which is distinctly different from those of either perilymph or endolymph. The anatomical appearance of this residue is consistent with a significantly higher protein content in the fluid filling the organ of Corti spaces, as suggested by Engstrom
[6]. (5) Backscattered SEM imaging was found to be an invaluable tool for th~~,evaluation of interior features of tissue samples and for the. detection of crystalline fluid residues. Freeze-drying is commonly employed to immobilize ions for :he later collection of samples from complex biological tissues [ 17]. This technique has the obvious advantage of eliminating ion movement due to fluid displacement or diffusion, greatly r,ducing opportunities for sample contamination. While in rive electrolyte-volume relationships of fluids are lost during freeze-drying~ the rdative proportions of electrolytes ca.a readily be measured by conventional elemental analysis teelmiques or by EDXA. The cochlea, almost completely enclosed in bone, is not an obvious c~didate for cryogenic freezing. It is perhaps for this reason that few studies have attempted to utilize freezeMrying to iramobilize ions in the cochlea. The difficulties pre~ented by the freeze-dried preparation of samples are many. Perhaps the most significant potential problem is the formation of large ice crystals during freezing. To minimize the size of ice crystals, investigators have used a variety of cryogenic fluids. Although extremely cold (-196°C), the heat transfer characteristics of liquid nitrogen make it an unsuitable heat sink. Organic compounds like Freon 12 or isopentane cooled ha liquid nitrogen have e ~ g rates o f - l ~ - I ~ ° C / s and have been used by many investigators [ 17]. Regardless of the c o o ~ g rate of the immersion bath, movement of heat out of a bioIogic~ specimen is limited by the relatively poor conduction characteristics of water, the primary biological so|ve~t. Thus it is difficult to ac~eve rapid
16 freezing in large biological ~ p l e s . In the ~ e a , the bony ~ ~ fu ILn~its heat " o transfer. However, s u b d i ~ o n of the coctflea or even t ~ ~ of f ,zb~e~ ~ ° would produce greater damage to ion cornpmmen ts than freeze artifact. ~ orgy ~ . able means ~f freezing the cochlea is to quench Me inner e2r w~hole, et at. [25] have estimated that freezing of the intact c.ochlea in Freon 12 cocked to -159°C is not complete until about 1.5 s after quenching. This freezing time undoubtedly a~ow~ ice G crystal formation in cochlear fluids and tissues. The uz~ o f ~ e 1¢ecrystals formed, ~ d the amount of physical damage c a r d , would d e ~ d upon the e ~ t f r e e ~ g time -topoint within the cochlea as weU as the protein and lipid ~ n t of the p ~ ~ fluid or tissue in question. The signifiem-lt questions for the use of freezing and subse t freezedrying in the analys/s of ion content of ¢ochle~~fluids and c , ~ is ~ degree to which ice crystal formation causes the m o v e ~ n t of ions out of the p~tions ~ they ~ g ~ y in rive. An ~spcct of ice crystal formation wMch could potentially alter ion c o m ~ t m e n t boundaries is the breakdown of ceil membr~e~ ~ d associated cetl-cgH junctions during " n , and then during intra~llular crys~l formation. ex,racellular crystal formatlo Fluid and tissue cooling, and thus ice ¢ryst~ formation, pt~.'~mab|y ~ s with ¢och. lear location. The most rapid cooling probably occurs close to ,he codflear bone, ~ ,pirM ligameni and stria vasoularis, and in perilymph. Cooling of more cgntr~ly located struc. tures such ~s the organ of Corti may be significantly slower. Oar anatomie.al ob~r~ltions suggest that heat loss may occur more rapidly in tissue surfaces adjacent to fl~d, ~ c e vesiculation of cytoplasm is smallest at this location. The slowest cooling ap~ars to occur at the center of large, contiguous cell groups like the Dieter's cells. Freezing of t ~ from fluid boundaries inward may limit the degree to which ice cryste~ls penetrate cell memb,anes, d'ae to smaller crystal size and possibly orientation of cry~al pl~me, parallel to men~brane surfaces. An important consequence of the apparent p:e~rvation of cochlear ~el[ membranes adjacent to fluid is the potential that fluid compartment integrity is maintained during freezing and freeze-drying. It seems likely that both intrac~ltular and extracellular ion distribution would be maintained if there were no major defects in the cell membrane. Even with small membrane discontinuities, it i.~ possible that movement of ions is limited to a small region around the membrane defect, ~ c e both tmu¢ and fluid would be in the process of freezing at the time of membrane penetration. ~n addition to ice crystal formation, another potential source of ion movement is the freeze-drying process itself. Even in a tissue matrix, in w~dch ions are probably bound to various protoplasmic constituents, only aepression of temperature to the eutectic point of water ( - 130°C) can absolutely suppress ion movement. Sublim~tio~ of water has been observed to cause slight movement of ions in tissue even when temperatures are main. rained below -80°C [4]. However, given the size and the bony capsule of the intact cochlea, the use of extremely low temperatures in freeze.drying is :~:,t practical. Whe~ water ~s removed at the moderatly low temperatures (-40°C) necessary for cochlear free~dryh~g, some ion movement is to be expected, especially in the case of fluid with a reintively low protein and lipid conlent such as endolymph or periiymph. |t is not surprish~g, therefore, that the contents of the cochlear scalae after freeze.drying are not uniform, ~hhfly.scattered ions. The degree to which ions condense into solid masses during freeze. drying reflects the size and configuration of proteins mad lipids w~ch migh~ form a matrix for the dehydrating electrolytes.
I7 Our
obse
that ion movement due to cell membrane puncture
U~g
repeated obsemtions on isolated fluid residues from the codflea, we have fuqat spectra of endolymph residue indicate high levels of potassium, somewhat lower levels of chlorine, and very low leveis of sodium. EDXA ,,~pectra obtained from perflymph residue show approximately equal levels of sodium and c~orine, with trace of ~dealcium,/The electrolyte content of the two major cochlear ~in by EDXA spectra are consistent with the best values which have been by other assays. ~ e extols of potassium over chlorine in endolymph has been in ~¢veral mammMian species [7,14 ~23,24]. The equal levels of sodium and c h l o ~ e which w~~.observed in perilym?h of the chindlla are similar to those seen in the eat [7,14,23] and ~ e the relative excess of sodium observed in the guinea pig [7,24]. The low levels of sodium in endol~zmph spectra and of potassium in perilymph spectra indicate no s ~ f i c a n t admixture of the fluids filling the three cochlear scalae during freezing or freeze.drying. Furthermore, mierodissection of freeze-dried inner ears has routinely provided samples of endolymph a~d perilymph with a high order of purity. This is in contrast to many ~tudies based upon invasion of the cochlear fluid spaces in a hydrated state [2,12-14,17]. We conclude chat the limitations of our methodology, such lo~ of absolute electrolyte-volume rela,ionships in fluid residues, are outweighed by the low levels of sample contamination which can routinely be achieved. EDXA spectra obtained from fluid residues at various cochlear locations have implications c o n e e ~ g the distribution and phyziology of endolymph. Our data provide strong evidence that the fluid which occupies the space between the rectorial membrane and the reticular lamina is endolymph. Flock [6] ri~easured chlorine and potassium levels in the subtectorial ~ace utilizing EDXA. He observed significant levels of potassium, and inter. preted this to reflect the presence of endd:Jmph. He did not, however, measure sodium levels. Ross [19] obtained EDXA spectra fr3m both sides of the rectorial membrane. She reported significant levels of sodium m s~ctra obtained from the underside of the membrane, in addition to potassium, and concluded tttat a fluid similar to perilymph must occupy the subteetorial space. In direct conflict with the data of Ross [ 19], our spectra. show equally high levels of potassium, aLd low levels of sodium, in fluid residues on both ~des of the rectorial membrane. In no inst~ce have we observed significant levels of sodi. um emi~on in spectra obtained from the ~eetorial membrane or from fluid residues associated with the membrane. EDXA ~ c t r a obtained from cochleal and utricular endolymph show equally Mgh pot a ~ u m and low ~dium content. It has beer~ suggested by several authors that the electrolyre compoation of ~ndolymph is different in the two divisions of the inner ear, based upon assays of fluid samples obtained by mieropipett¢ penetratiom of the endolymphatic compartment [18,21|. Separate origins and circulations of cochlear and vestibular endolymph were also p r o I ~ d . Our data do not support a difference between endolymph in the two divisions of the labyrinth. Differences reported in previous investigations may represent the difficulty of obtaining uncont~rlinated samples of endolymph from the sac"_fhe residues of Lrmer ear fluids were distinct in the~ anator~ica] appearance, presumably becau~ of differen~s m their protein and ~pid content, as well as *_hesize and
18
distribution of ice c r y ~ produced during freez~g. P e v 3 ~ ~ n t P y e ~ b i t e d the greatest degree of condensation durizlg f r e e z e ~ i n g , frequently ~ in scab tymp~i and vestibuli empty of fluid residue. ~ ~ e s t s a rels low pr ~ m or lipid content. Endolymph, in c o n t r ~ , ~.ally precipitatedas an ~ributed, cottony mass which completely ~ the sca~ media. ~ ~ a higher protein or lipid content than perilymph. Comparison of EDXA spectra obtained from the two fluids shows that while endolymph spectra c ~ t e n t l y show m e a s u m b ~ ~ o n sulfur window, suggesting protein, con~derably ~ ~ o n in ~ w i n ~ ~ observed in purllymph spectra. This observation is not c o ~ t with most previous e~imates of the prorein content of endolymph and pe~ymph, in w ~ ~ latter has been ~own to exhibt a higher protein content [7,16~23]. However, these observations were ~ performed with micropipette sampling. Tonndorf et el. [26] and Rauch [15] have p r e s e n ~ anatomical evidence that a matrix of glycoprotein is present in scala media. Such a matrix might not be sampled during withdrawal of fluid through a small aperture pipette, but would certainly be included in our micro-dissected ~mples. Our data may reflect the presence of s, ch a glycoprotein in scala media. Both pertlymph and endolymph displayed characteristic crystals ~s part of their fluid residues, The appearance of cryst~s as refidues of freeze-drying has no~ aiwsys been reported from the cochlea. Flock [6] described the fluid residues as cottony muses of filaments or plates. Th~ma~n et al. [25] mentioned fluid residues but did not describe them. However, Peterson et al. [ 14] observed crystals m freeze.dried droplets of undolymph and perilymph which had previously been withdrawn from the inner ear in micropipettes. It seems possible that the failure of some investigators to report crystals in cochlear fluid residues resulted from the lack of ability to detect high-denfity particle.s embedded in the residue matrix. We have found backscattered imaging to be the only reliable means of identifying crystal presence ~ fluid residues or on tissue surfaces. The ~ack of crystalline fluid residues in ~ e organ of Corti ~paces indicates that the fluid which normally occupies the spaces may have a higher protein or lipid content than either perilymph or endolymph. It seems unlikely that the restricted size of the fluid spaces in the organ would restrict crystal formation, since cryst~ were ~way$ abundan t in the subtectorial space and inner sulcus. Engstrom [5] has suggested that the protein content of 'cortilymph' maybe higher than that of the other cochlear fluids, on anatomical grounds. High protein content is also characteristic of e x t ~ H u l a r spaces with restricted fluid circulation. This might well apply to the spaces of the organ of Corti. The data obtained in the present investigation leave many important questions concerning cochlear ion distribution, and the effects of freeze.drying upon that distribution, unanswered. While it seems clear that there is no gross movement of ions between the central regions of the cochlear scalae, we have not determined the degree to which ions may move on a smaller scale. This would include ion movement from one cell to another, as well as from the interiors of cells ~o the edges of fluid compartments. T1t$ extent of such movement is of particular importance to the measurement of ion distribution in the fluid spaces of the organ of Corti, as wall as in cochiear t~ues. The variability which we have observed m EDXA measurements of element content has been quite low. This suggests that more quantitative use of E D X A is feasible in the inner ear, providing that appropriate controls are employed. We are currently exploring
19
the rive us~ of EDXA, to characterize ch~ges in cochlear ion distribution under raeh conditions as noise exposure, drug treatment and anoxia. REFERENCES [1] Bosher, S.~-. and Warren, R.L. (1968): Observations on th~ electrochemistry of the cochlear endolymph of th, rat. Proc. R. Soc. London 171,227-247. [2] Citron, L., Exley, D. and Hallpike, C.S. (1956): Formation, circulation and chemical properties of labyrintl2ine fluids. Br. Med. Bull. 12, 101-106. [3] Citron, L. and Exley, D. (1957): Recent work on biocllemistry of the labyrinthine fluids. Proc. R. Soc. Med. 50, 697-701. [4| Dorge, A., Rick, A., Gehfing, K., Mason, J. and Thurau, K. (1975): Preparation and applicability of freeze-dried sections in the microprobe analysis of biological soft tissue. J. Microsc. 22, 205214. [5] Engstrom, H. (1960): Corfilymph. The third lymph of the inner ear. Acta Morphol. Neerl. Scand. 3, 192-204. [6] Hock, A. (1977): Electron probe determination of relative ioI~ distribution in the inner ear. Acta OtolaryngoL 83, 239-244. [7] Johnstone, C.F., Schmidt, R.S. and Johnstone, B.M. (1963): godium and potassium in vertebral cochlear endolymph as determined by flame microspectropho :ometry. Comp. Bioehem. Physiol. 9, 335-358. [8] 3uhn, S.K. (1971): Perilymph findings in experimentally indue :d acidosis. Arch. Otolaryngol. 93, 384-387. [9] Lawrence, M. (1967): Elec~:~cpolarization of the tectorial metnbrane. Ann. Otol. Rhinol. LaryngoL 76, 287-312. [10] Lawrence, M., Nuttal, A. aad Clapper, M. (1975): Electric~l potentials and fluid boundaries within the organ of Corti. J. Acoust. Son. Am. 55,122-1~8. [I1] Lira, D.J. (1972): Fine morphology of the teetorial membrare; its relationship to the organ of Corti. Arch. Otolaryngol. 96, 199-215. [12] Miyake (1960): Biochemical study of labyrinthine fluids. J Otorhinolaryngol. Soc. 3ap. 63, 1-18. [13] Oliveim, C.A., Juhn, S.K. arid Kim, C.S. (1077): The effect of microembolization of cochlear capillaries on the ionic composition of perilymph and endolymph. Trans. Am. Aead. Ophthalmol. OtolaryngoL 84, ORL 9C8. [14] Peterson, S.K., Frishkopf, L.S., Lechene, C., Oman, C.M. and Weiss, T.F. (1978): Element corn-" position of inn~ ear lymphs in cats, lizards, and skates determined by electron probe microanalysis of liquid samples. J. Comp. Physiol. 126, 1-14. [15] Ranch, S. Unpublialed observation, cited in Tonndorf et al., op. tit. [16] Rauch, S. (1964): Biochemie des Hororgans: Einfiihrung in Methoden und Engebnisse. G. Thieme Verlag, Stuttgart. [ 17] Robards, A.W. (1974): Ultra~tructural methods for looking at ceils. Sci Prog. Oxford 61, 1-80. [18] Rodgets, K. and thou, LT.Y. (1966): Concentrations of inorganic icns in guinea pig inner ear fluids. 1: Concentration of potassium and sodium in cochlear and utricvlar endolymph. J. LaryngoL 80, 778-790. [ 19] Ross, M.D. (i974): The rectorial membrane of the rat. The Am. J. Aria ~.. 139,449-4:32. [20] Schuknecht, H.F. and McNoiU, R.A. (1966): Light microscopic observ ltions on the pathology of endolymph. 3. LaryngoL 80, t - 1 0 . [21] Setlick, P. and 3ohnstone, B. (1975): Production and role of inner ea::~fluid. Prog. Neurobio]. 5, 1-18. [22] Seymour, 3.G. (1954): Ob,ervations on the circulation of the cochlea. 5. LaryngoI. 68,689-697. [23] Silverstein, H. and Griffin, W.L. (1970): Compaxi~n of inner ear flui is in the antemortem and po~mortem state of the cat. Ann. Otol. Rhinol. Laryngol. 79, 178-t86.
20 [24 | Smith, C ~ . , L ~ O~H. ~ W La~Tngo~cope ~ , ~ A I - I ~ [25] Th~llman, Ro, Comeg'/s, T.H. and fr~z~-dri~ time for u[ttan~ictoch [26] Tonndoff, J., DuvaH, A..I. and Re~ various vita~ ~tain~. Ann. OtoL RhinoL ~ ~ o ~ .
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