organic material concentration in auditory outer hair cells measured by laser interferometry

6
0 1995 Wiley-Liss, Inc. Cytometry 2O:l-6 (1995) Organic Material Concentration in Auditory Outer Hair Cells Measured by Laser Interferometry E. Laffon, D. Dulon, C. Aurousseau, S. Dilhaire, and W. Claeys Laboratoire d’Audiologie Experimentale,Universite de Bordeaux 11, H6pital Pellegrin, Bordeaux (E.L., C.A., D.D.), 1.U.T. A, Dkpartement de Mesures Physiques, domaine universitaire, Talence (E.L), C.P.M.O.H. and Laboratoire de Micrdlectronique I.X.L., UniversitC de Bordeaux I, Talence (S.D., W.C.), France Received for publication June 17, 1994; accepted November 1, 1994. Outer hair cells (OHC) of the mammalian cochlea are quasicylindrical cells of Merent length, which play a major role in hearing at threshold. Their par- ticular shape allows the use of a noninvasive laser interferometric technique of isolated cells in vitro in order to measure the organic material concen- tration (OMC), hence the density of each cell body. In most (95 %) of the OHCs isolated from the same guinea pig, when the cell diameter is normalized, the results show that the cell body OMC does not vary with cell length. In different animals, the re- spective normalized OMC mean values can vary be- tween 70 kg/m3 and 103 kg/m3. A few OHCs with morphological particularities often possess cell body OMCs > 103 kg/m3. The results of the interfer- ometric measurements in isolated OHCs confirm that density variations in the cell bodies are not in- volved in a sound fiequency coding. The in vitro OMC variations of the OHCs could be related to the isolation procedure; however, they could also cor- relate with actual in vivo OMC variations. 0 1995 Wiley-LiS~, I~c. Key terms: Isolated cell interferogram, density in cells, cochlea Mammalian sound frequency detection and amplifica- tion at threshold of hearing are still not clearly under- stood. It is thought that an active cochlear process selec- tively amplifies the vibrations of the basilar membrane in the cochlea (8). Among the different cells composing the organ of Corti, the outer hair cells (OHCs) are likely responsible for this mechanism ( 19). Schematically, mammalian OHCs are quasicylindrical cells of nearly constant 10 pm diameter, and of length ranging between 30 Fm and 80 pm in the guinea pig (9,lS). Their apical pole is formed by a cuticular plate with a ciliary bundle, and the cell nucleus is in basal position (Fig. 1). In the organ of Corti, the OHCs are held at both ends between the body and the phalangeal process of supporting cells (Deiters cells). In vitro, the isolated OHCs show unique electro-mechanical contractions along their axis, in re- sponse to electrical stimuli in the audio frequency range ( 1,12,15).However, although an electrical resonance has been evidenced in turtle hair cells (1 l), until now no resonant character of this fast cellular electro-motility has been directly evidenced in mammals. Direct mechan- ical stimulations of the OHC lateral wall produce a sharply tuned slow motile response (5). Furthermore it has been recently reported that such a stimulation can also induce phasic cellular length changes,which suggest a damped forced mechanical resonance (6). The mammalianOHC length could be one parameter of an intrinsic sound frequency coding (9,18). In a previous work (16), we have shown that another basic mechanical parameter,the cell body density,did not vary significantly versus length. These results suggested that variations of cell body densitywere not involved in a kequency coding, unlike for length. The experiments, which were per- formed by optical trapping of OHCs isolated from many guinea pigs, have, however, revealed important variability in the OHC masses for each cell length. The aim of the present experiments is to characterize in detail, in isolated OHCs in vitro, the density variations of the cell body. Furthermore, the results provide addi- tional information about a fundamental mechanical pa- rameter for modeling the “active” nonlinear cochlea ( 13). The tubular shape of the OHCs allows experiments to be performed by means of a noninvasive laser inter- ferometric technique. The method measures locally the refractive index difference between the cell body and the surrounding medium. Then, when the cell diameter is normalized, we can calculate a local normalized organic material concentration (OMC) and a normalized density. MATERIALS AND METHODS Cell Preparation and Selection Pigmented guinea pigs (250-350 g) with a positive Preyers’ reflex are decapitated after being deeply anaes- thetized, the temporal bones quickly removed, and the ~ ~ ~~ Address reprint requests to Dr. Eric Laffon, Laboratoire dAudiologie Experimentale, H6pital Pellegrin, 33076 Bordeaux Cedex, France.

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0 1995 Wiley-Liss, Inc. Cytometry 2O:l-6 (1995)

Organic Material Concentration in Auditory Outer Hair Cells Measured by Laser Interferometry

E. Laffon, D. Dulon, C. Aurousseau, S. Dilhaire, and W. Claeys Laboratoire d’Audiologie Experimentale, Universite de Bordeaux 11, H6pital Pellegrin, Bordeaux (E.L., C.A., D.D.), 1.U.T. A,

Dkpartement de Mesures Physiques, domaine universitaire, Talence (E.L), C.P.M.O.H. and Laboratoire de Micrdlectronique I.X.L., UniversitC de Bordeaux I, Talence (S.D., W.C.), France

Received for publication June 17, 1994; accepted November 1, 1994.

Outer hair cells (OHC) of the mammalian cochlea are quasicylindrical cells of M e r e n t length, which play a major role in hearing at threshold. Their par- ticular shape allows the use of a noninvasive laser interferometric technique of isolated cells in vitro in order to measure the organic material concen- tration (OMC), hence the density of each cell body. In most (95 %) of the OHCs isolated from the same guinea pig, when the cell diameter is normalized, the results show that the cell body OMC does not vary with cell length. In different animals, the re- spective normalized OMC mean values can vary be- tween 70 kg/m3 and 103 kg/m3. A few OHCs with

morphological particularities often possess cell body OMCs > 103 kg/m3. The results of the interfer- ometric measurements in isolated OHCs confirm that density variations in the cell bodies are not in- volved in a sound fiequency coding. The in vitro OMC variations of the OHCs could be related to the isolation procedure; however, they could also cor- relate with actual in vivo OMC variations. 0 1995 Wiley-LiS~, I~c.

Key terms: Isolated cell interferogram, density in cells, cochlea

Mammalian sound frequency detection and amplifica- tion at threshold of hearing are still not clearly under- stood. It is thought that an active cochlear process selec- tively amplifies the vibrations of the basilar membrane in the cochlea (8). Among the different cells composing the organ of Corti, the outer hair cells (OHCs) are likely responsible for this mechanism ( 19). Schematically, mammalian OHCs are quasicylindrical cells of nearly constant 10 pm diameter, and of length ranging between 30 Fm and 80 pm in the guinea pig (9,lS). Their apical pole is formed by a cuticular plate with a ciliary bundle, and the cell nucleus is in basal position (Fig. 1). In the organ of Corti, the OHCs are held at both ends between the body and the phalangeal process of supporting cells (Deiters cells). In vitro, the isolated OHCs show unique electro-mechanical contractions along their axis, in re- sponse to electrical stimuli in the audio frequency range ( 1,12,15). However, although an electrical resonance has been evidenced in turtle hair cells (1 l), until now no resonant character of this fast cellular electro-motility has been directly evidenced in mammals. Direct mechan- ical stimulations of the OHC lateral wall produce a sharply tuned slow motile response (5). Furthermore it has been recently reported that such a stimulation can also induce phasic cellular length changes, which suggest a damped forced mechanical resonance (6).

The mammalian OHC length could be one parameter of an intrinsic sound frequency coding (9,18). In a previous work (16), we have shown that another basic mechanical

parameter, the cell body density, did not vary significantly versus length. These results suggested that variations of cell body density were not involved in a kequency coding, unlike for length. The experiments, which were per- formed by optical trapping of OHCs isolated from many guinea pigs, have, however, revealed important variability in the OHC masses for each cell length.

The aim of the present experiments is to characterize in detail, in isolated OHCs in vitro, the density variations of the cell body. Furthermore, the results provide addi- tional information about a fundamental mechanical pa- rameter for modeling the “active” nonlinear cochlea ( 13). The tubular shape of the OHCs allows experiments to be performed by means of a noninvasive laser inter- ferometric technique. The method measures locally the refractive index difference between the cell body and the surrounding medium. Then, when the cell diameter is normalized, we can calculate a local normalized organic material concentration (OMC) and a normalized density.

MATERIALS AND METHODS Cell Preparation and Selection

Pigmented guinea pigs (250-350 g) with a positive Preyers’ reflex are decapitated after being deeply anaes- thetized, the temporal bones quickly removed, and the

~ ~ ~~

Address reprint requests to Dr. Eric Laffon, Laboratoire dAudiologie Experimentale, H6pital Pellegrin, 33076 Bordeaux Cedex, France.

2 LAFFON ET AL.

FIG. 1. Mammalian cochlear outer hair cell. An isolated mammalian cochlear OHC in vitro is a quasicylindrical cell of -10 Fm diameter. In the guinea pig, its length ranges approximately between 30 pm and 80 pn coding for different sound frequencies. The cell exhibits a basal nucleus and an apical cuticular plate with a ciliary bundle.

bulla and bony walls of the cochlea immediately opened. After removal of the stria vascularis and the tectorial membrane, the different turns of the organ of Corti are separated from the spiral lamina with a thin metal probe. The dissected strips of the organ of Corti are transferred into a solution of collagenase medium (type IV from Sigma; 0.5 mg/ml) and incubated for 30 min at room temperature. The strips are then removed from the col- lagenase medium to a 100-yl drop of culture medium, where the dissociation procedure is mechanically achieved by gentle flux and efflux of the tissue pieces with a Gilson 100 pipette. Isolated OHCs are finally trans- ferred into a chamber filled with culture medium, whose bottom has been previously metal coated in order to cre- ate a mirror, and 30 min later, the cell attachment to this surface is sufficient to place the chamber vertically in the experimental setup, after closing the chamber with an optical quality glass slide (0.45 mm thickness). Both iso- lation procedure and experiments are performed in Hanks' Balanced Salt Solution (HBSS)( 16).

We choose the OHCs to be analyzed as cylindrical as possible, and without light microscopic heterogeneities at the level of the cytoplasm of their cell body. Cells that present morphologic alterations such as cytoplasmic granulations, irregular outlines, or swelling are not stud- ied. However, measurements are performed in a few ab- normal O H 0 that do not present such alterations, but whose nucleus is partly or entirely positioned in the me- dial third of the cell body (17).

Interferometric Experimental Setup The experimental setup (7) is sketched in Figure 2. It

is basically the well-known Michelson interferometer. The laser source is a single-mode, polarized He-Ne laser whose wavelength (A) is equal to 0.6328 ym. Using a

beam-splitter (BS), the laser beam is split into a reference beam and a probe beam illuminating the sample. A lens is placed before the beam-splitter in order to expand the laser beam. In the probe arm of the interferometer, a microscope objective ( X 20, NA:0.3, WD:17 mm) al10WS complete visualization of a 80-pm-long OHC, with the light source when the laser is shut off. When the light source is shut off and the laser probe beam opened, the focusing of the expanded beam by the microscope objec- tive is limited, and the cells are entirely illuminated with a quasiplanar wave. A similar microscope objective is also placed in the reference arm of the interferometer, be- cause the two interfering beams captured by the CCD camera must possess similar characteristics to get high contrast interference fringes. After the microscope objec- tives, the radius of the expanded beams is -50 pm and the light intensity is <ZOO pW. Different interference fringe patterns can be obtained with the X,Y translation stage of the reference microscope objective.

OHC Interferogram In our experiments, the reference and probe laser

beams of the Michelson interferometer are projected on the CCD camera with an angle between them, and a lin- ear interference fringe pattern is observed. The spacing of the fringes depends on the angle, and their position de- pends on the respective optical path lengths travelled by the beams. Thus, e.g., if a glass slide is placed in the path of one beam, the entire interference pattern will be shifted without a variation of the fringe spacing. This spacing can be taken as unit for the measurement of the pattern shift. When the glass slide induces a variation of the optical path length equal to the laser wavelength, the shift is equal to the unit of the instrument.

In our experiments, the vertical test chamber is placed perpendicularly to the probe laser beam. The laser light reflection occurs just behind the cell body, which is linked to the metal-coated surface of the chamber. The laser light passes through the cell twice. The refractive index difference between the cell body and the HBSS medium (kHC - nHBSS) modifies the optical path of the coherent probe light at the place of the cell. After inter- ference with the reference beam and when the linear interference fringe pattern is positioned perpendicularly to the OHC axis, the correlated local shift of the interfer- ence pattern leads to the cellular interferogram (Fig. 3). On the axis of a cylindrical cell of radius R (in pm), the variation of the optical path length is equal to 2.( 2R).( nOHC - nHBss). Thus the ratio of the correlated fringe shift (Ap) over the fringe pattern step (p) is equal to:

Ap/p = 4.R. (q,HC - nHBs)/X

Measurements of the Ap/p ratio are achieved on inter- ferograms, which are recorded with a video camera (Sony) on a U-Matic Sony VCR (VO-5800 PS). The images are analyzed with the image analysis software Image-1 (Universal Imaging Corp., West Chester, PA), using the

INTERFEROMETRIC MEASUREMENTS OF CELLULAR DENSITY 3

microscope objective

(x, y) test chamber I and probe mirror

LENSES

I I microscope translation

Video objective

lens +I+ stage

I

Laser E!l FIG. 2. Schematic diagram of the interferometric experimental setup

In the longest OHCs, light microscopic observations sometimes reveal dense axial structures, extending from the apical cuticular plate towards the basal nucleus. Since equation (1) supposes no refractive index heterogene- ities in the cellular cylinder, we carry out the interfero- metric measurements just above the basal nucleus, where there are no such dense structures. In the preceding equation, the cell sagital radius (R) is assumed to be equal to the cell frontal half width, which is the only dimension that we are able to measure in our experi- ments. In order to fit that assumption, no measurements are done when the shape of the fringe shifts is more rect- angular than semicircular.

FIG. 3. OHC interferogram and principles of analysis. The interference fringe shifts reveal a cylindrical OHC. The amplitude of a fringe shift in comparison with the fringe spacing can be measured in order to calcu- late a local cell body OMC (equations I and 11). A first averaged line intensity scan perpendicular to the linear interference fringes is made outside the OHC (grey curve). Then, a second line intensity scan is made along the axis of thc cell (white curve). The two scans bcgin below (or above) the cell in order to put them in phase and then to measure the interference fringe shifts in comparison with the respective fringe spac- ings, at different places of the ccll body.

function “Line Intensity Scan” after a X 2 zoom. Such an analysis is illustrated in Figure 3. Generally, three values of the Ap/p ratio give us a mean value and a standard deviation.

Normalized Organic Material Concentration and Density

The relation between refractive index and dry mass of cells was studied in the 1950s by Barer and Joseph (2,3). Barer has also established the principles of cellular inter- ferometry (4). The refractive index of a solution of pro- tein increases linearly with increasing the concentration. A living cell can be considered as a water solution of mainly proteins but also lipoproteins and aminoacids; thus its refractive index can be taken as a measure of the total amount of organic solids present. Barer and Joseph estimated the value of a coefficient, called the refractive increment (2), connecting the cellular organic material concentration (C, here expressed in k@m3 of cytoplasm) and the refractive index difference between the cell and the surrounding medium:

4 LAFFON ET AL.

(noHc - nHBss) = 0.00018 . C

Moreover, they demonstrated (3) that the cell body den- sity (db) can be estimated to:

d, = 1 + Cl4000 (3)

Before comparing the organic material concentrations (hence densities) in OHCs, it is necessary to make some adjustments. Indeed, since the diameter of an isolated cell ranges generally from 6 pm to > 11 pm in vitro, we have to normalize the OMCs relatively to this parameter in order to compare cell body OMCs. The normalized cel- lular diameter will be taken equal to 10 pm; see (9,18). The amount of organic materials per cell length unit in a given cylindrical cell body is equal to C.n.R2. Assuming that this amount does not vary significantly whatever the cellular diameter changes occuring during the cell isola- tion procedure and using equation (2), we can calculate a normalized cell body OMC (C,):

C, = c.(2R/10)2 ( 4 )

The OHC shape is not quite cylindrical, nor is it that of a prolate spheroid. Note that in each case the normaliza- tion leads to the same equation ( 4 ) . The cell length can- not be normalized because a cell length of reference is unknown. Therefore, it is taken arbitrarily equal to the measured length under light microscopy. In order to re- duce errors relative to this choice in the comparison of cell body OMCs, only OHCs with diameters measured between 6 pm and 1 1 pm are considered in these exper- iments, since an enlarged cellular diameter is generally accompanied by a cell shortening (10).

RESULTS Normalized Cell Body OMC of Isolated OHCs in

Guinea Pig With criteria of OHC selection defined under Materials

and Methods, we have only analyzed 1040% of the iso- lated cells in each preparation in vitro. For eight guinea pigs, we have measured the OMC of 74 isolated OHCs at the level of each cell body. Normalized cell body OMC vs. OHC diameter are presented in Figure 4. Most of the normalized OMCs are randomly distributed between 55 kgm’ and 105 kgm’, whatever the cell diameter ranging between 6 pm and 11 pm. This confirms the validity of our normalization. The relative errors of the individual measurements, due to the evaluations of the OHC diam- eter and of the Ap/p ratio, range generally between 8 and 15.5%.

Normalized Cell Body OMC of Isolated OHCs in One Individual Guinea Pig

The normalized cell body OMCs of living OHCs iso- lated from the same animal are presented vs. cell length in Figure 5. Whatever the cell length, the normalized cell

FIG. 4. Normalized organic material concentration of isolated OHC cellular body versus cell diameter, from eight guinea pigs. The concen- trations are homogeneously distributed for cell diameters ranging be- tween 6 and l l pm; this validates the normalization.

body OMC varies around a mean value; here, 70.9 kg/m3. Figure 5 demonstrates that a mean value of the normal- ized cell body OMC can be calculated for a given animal, whatever the length of the analyzed cells.

M e a n Normalized Cell Body OMC of Isolated O H 0 in Different Guinea Pigs

Comparison is shown in Figure 6 among the respective mean normalized cell body OMCs of isolated OHCs in eight guinea pigs. In seven animals, the mean normalized OMC does not differ significantly: <OMC> = 72.5 * 10.8 kgm’, 70.4 * 13.5 ks/m3, 72.6 2 18.5 kg/m3, 73.4 * 15.2 kg/m’, 70.9 * 10.1 kg/m3, 83.3 2 7.7 kg/m3, 86.8 13.3 kgm3 (n = 5, 5, 5, 14, 13, 9, 8). Only the mean normalized OMC of the animal H is significantly different: <OMC> = 102.3 2 12.4 kg/m3 (n = 15).

OMC Measurements in Isolated OHCs with Atypically Positioned Nucleus

As mentioned by Lim (17), abnormal OHCs whose nu- cleus is not placed in the basal third of the cell body can be observed in isolated cell preparations. In our experi- ments, these cells represented 0-7% of the total number of isolated OHCs in one individual animal. Most of these cells were longer than 60 pm. We analyzed interfero- grams of 13 atypical OHCs that met all the selection criteria defined in Materials and Methods. The OMC mea- surements were performed below the nucleus at the basal part of the cell.

For each guinea pig, the normalized OMCs of these atypical cells are found to be larger than the respective normalized OMC mean value. Seven over the 13 atypical analyzed OHCs possess normalized OMC values equal or above 110 kgm’. The highest measured normalized OMC (201 f 28 kg/m’) is for the OHC shown in Fig- ure 7 .

INTERFEROMETRIC MEASUREMENTS OF CELLULAR DENSITY 5

FIG. 5. Normalized organic material concentration of isolated OHC cellular body vs. length in one individual guinea pig. The normalized cell body OMC of the OHCs does not vary significantly with cell length.

- m

x D 0 100 n L -

80 u 0 I

60 ’L 0

V

5 40 D N ._ -

20 E

6 0

C

A B C D E F G H -= Guinea pig # c

FIG. 6. Mean normalized organic material Concentration of isolated OHC cellular body in different guinea pigs.

DISCUSSION In one individual guinea pig, the normalized cell body

OMC of most (95% ) of the OHCs does not vary with cell length (Fig. 5). Thus, Figure 5 confirms that sound fre- quency coding does not involve variation in cell body density, unlike what is suspected for length (18). This confirms previous results obtained with OHC optical trapping (16), which gave an estimation of the normal- ized cell body density, d,, between 1.01 1 and 1.017 (mean value 1 .0 14). In the present experiments, equation 3 leads to a mean normalized cell body density that is slightly larger: d, = 1.020 k 0.004. But we must take into account a 5% relative uncertainty due to the estimation of the refractive increment (2) and an error on the pro- teic specific volume (unknown). One could also explain the slight discrepancy between the two techniques by considering that laser trapping is a much more invasive technique than laser interferometry: power, beam diam-

FIG. 7. Outer hair cell and the bent phalangeal process of a supporting cell (Deiters cell) interferograms. This particular OHC, whose nucleus is atypically positioned in the medial third of the cell body, possessed the largest measured OMC.

eter, and wavelength ratios between the two experiments are -60/0.2 (mW), 8/100 (pn) , and 514/633 (run), re- spectively. Furthermore, in the trapping method the den- sity estimation has been established with the assumption that the cuticular plate and nucleus mass don’t vary sig- nificantly with cell length, and the normalization we used underestimated the cell body density (the OHC shape is nearly but not strictly cylindrical).

The respective measurements we have performed in each guinea pig show that whatever the cell length, the normalized cell body OMC of the isolated OHCs could vary by 10-25% around a mean value (Fig. 6). Such vari- ations must be compared with individual measurement relative errors ranging between 8% and 15.5%. Further- more, the comparison of normalized OMC mean values in different guinea pigs suggests that the cell body normal- ized OMC mean value could be different from one animal to one another, ranging between 70 and 103 kg/m’. Both these variations correlate with the variation in the masses of isolated OHCs, whatever the cell length, which were observed with optical trapping (16). A possible hypoth- esis that could explain these variations involves the cell isolation procedure, which could affect the OHCs differ- ently. Isolation is a complex procedure and “standard’ OHCs cannot be obtained for a given cochlea.

However, can in vitro OMC variations of the cell body of isolated living OHCs actually be correlated with in vivo variations? In T.E.M. experiments, lurato (14) con- cludes that “in many types of pathology, spontaneous or experimental, there are variations from one place to an- other along the basilar membrane.” In our laser interfer- ometric experiments, the additional measurements in iso- lated cells presenting an atypical positioned nucleus could support actual in vivo OMC variations. These ab- normal cells often possess large normalized OMCs, which

6 LAFFON ET AL.

are unlikely to correlate with OMC variations caused by the isolation procedure. Indeed, if the isolation proce- dure induces a nucleus rising toward a medial position in the OHC, this is unlikely to occur without an important cell alteration, and then without an important loss of or- ganic materials. The consequences of actual in vivo cell body OMC variations in OHCs could be taken into con- sideration for the micromechanical properties of the co- chlear partition. Using both laser trapping and laser in- terferometry results, we have calculated that a 30% variation of the cell body density of a 60-pm-long OHC leads to about a 20% variation of its total dry mass.

ACKNOWLEDGMENTS The authors are very grateful to J.-M. Aran for his sup-

port and discussion during the study. They also thank J.-H. Godard, A. Guilhaume, and V. Quintard for helpful discussions and technical assistance. They acknowledge J. Schacht for comments of the manuscript. This work was supported by I.N.S.E.RM., Conseil Regional d'Aquitaine, and C.R.E.D..

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