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Alcian Blue staining of cochlear hair cell stereocilia and other cochlear tissues
Hearing Research, Elsevier
153
23 (1986) 153-160
HRR 00792
Alcian Blue staining of cochlear hair cell stereocilia and other cochlear tissues Peter A. Santi * and Craig B. Anderson Department
of Otolaryngolo~,
Unruersity of Minnesota Medical School, Mmneapolw (Received accepted
MN 55455, U.S.A.
9 December 1985; 29 January 1986)
Using whole-mount surface preparations of the chmchilla organ of Corti. the anionic characteristics of cochlear tissues was investigated with the polycationic dye Alcian Blue. Our primary goal was to specifically stain the hair cell stereocilia for the purpose of producing cytocochleograms. To specifically stain hair cell stereocilia we investigated the effects of different fixatives and the ‘critical electrolyte concentration’ [Scott and Dorling (1965) Histochemie 5, 221-2331 of MgCl, on staining specificity. The stereocilia and the tectorial membrane were the most intensely stained cochlear structures and presumably have a strong negative charge. The most important factor in producing specific staining was fixation in osmium tetroxide rather than the addition of an electrolyte to the Alcian Blue staining solution. The use of Alcian Blue produced intense staining of hair cell stereocilia against an unstained background using standard, brightfield light microscopy Alcian
Blue. cochlea,
hair cells, stereocilia
Introduction Alcian Blue 8GX (AB) is a soluble form of copper phthalocyanine which contains up to 4 positively charged isothiouronium groups per molecule (Scott et al., 1964). The mechanism of AB staining has been elucidated by Scott and co-workers (1960, 1964, 1965) and will be briefly reviewed. Due to its cationic nature, AB is strongly held in the negative electrostatic field associated with glycoconjugate polyanions such as chondroitin sulfate, hyaluronic acid, heparin and acid glycosaminoglycans. The majority of the glycoproteins and glycolipids of the cell are associated with the surface membrane and extend into the external environment (Glick and Flowers, 1978). Staining of polyanions is completely blocked by prior treatment with cetyl pyridinium chloride, which is a cationic detergent. The precipitates formed by polyanions with AB are soluble only in strong salt * Present address: S.E.. Minneapolis, 037%5955/86/$03.50
Research East, 2630 MN 55414, U.S.A.
University
Avenue
cb 1986 Elsevier Science Publishers
solutions. The addition of an electrolyte, particularly MgCl 2, to an AB staining solution has been shown to displace the dye from specific binding sites. The basis of this phenomenon is competition between the cations of the electrolyte and the isothiouronium groups on AB for the negative sites on the polyanion. The concentration of salt required to dissolve a given complex depends upon the relative affinities of the competing cation for the polyanion. The ‘critical electrolyte concentration’ (CEC) for several specific negative sites on polyanions has been determined (Scott, 1960; Scott and Dorling, 1965). The anions from the electrolyte added to the staining solution also affect staining intensity. By the blocking of 1 or more of the 4 cationic sites on the AB molecule, more dye is required to combine with the same number of tissue polyanions and the staining appears more intense. The presence of acid glycosaminoglycans (AGAG) in cochlear tissues has been known since the time of Belanger (1953) who reported the presence of sulfated compounds in the tectorial
B.V. (Biomedical
Division)
154
membrane. The AGAG are present on ceil membranes and have a strong negative charge due to the presence of sulfate ester groups and/or carboxyl groups of uranic acids (Scott and Dorling, 1965); thus, they bind cationic dyes. Most of our present knowledge concerning the distribution of AGAG in inner ear tissues is derived from the work of Saito (1982). Using biochemical methods, he found that the distribution of AGAG in cochlear tissues was: 0.1% in the tectorial and Reissner’s membrane, 0.6% in the stria vascularis and spiral ligament and 0.5% in the basilar membrane, spiral limbus and organ of Corti (concentration expressed as: AGAG/dry wt. tissue). However, using histochemical methods involving staining with periodic acid Schiff’s reaction, colloidal iron, methylation and hyaluronidase digestion, neutral red and AB (acidified) Saito (1982) found that AGAG distribution was strongest in the basilar and tectorial membranes and negative in the stria vascularis. Ruthenium red, which is an electron dense, cationic dye also has a strong affinity for the tectorial membrane (Tachibana et al., 1978) and for the hair cell stereocilia (Slepecky and Chamberlain, 1985). The soft-surface preparation method of Axelsson et al. (1975) produces whole-mount preparations of the organ of Corti, which are relatively easy to dissect (compared with plastic embedded whole-mounts), without the loss of significant portions of the basilar membrane. However, these preparations are only lightly and evenly stained by the osmium tetroxide (0~0,) fixative and thus recognition of cochlear cells is usually done using phase or interference microscopy. Since recognition of the hair cell stereocilia is one of the most important criteria in assessing the presence or absence of a hair cell for producing a cytocochleogram, a specific stain for these structures would be desirable. Katagiri et al. (1968) described a method of staining hair cell stereocilia and other cochlear structures using a silver method. However, silver staining methods are known for their capriciousness. In this paper, we describe a method of reliably staining hair cell stereocilia with AB which is compatible with the soft-surface preparation method of Axelsson et al. (1975).
Methods Twenty chinchillas, approximately 1 year old. were used in this study. The animals were anesthetized with an i.p. injection of sodium pentobarbital (60 mg/kg). They were decapitated using bone forceps and scissors. The bullas were separated from the skull by first cutting through the bone between the bullas with scissors. The bullas were then removed by inserting thumbs into the incision and applying upward pressure on the base of the bullas with the index fingers. The bullas were carefully opened to expose the cochleas and the stapes footplate was dislodged from the oval window. A small hole was hand drilled, with a sharpened pick, through the otic capsule of the Scala tympani beneath the first cochlear turn. Several different fixatives were used for this research. Formalin was prepared from paraformaldehyde as a 4% solution in phosphate buffer (pH 7.2). Glutaraldehyde was obtained from Polysciences as a 10% aqueous solution and diluted with phosphate buffer until it was 2% glutaraldehyde in 0.1 M phosphate buffer. 0~0, was also obtained from Polysciences as a 4% aqueous solution and diluted to the appropriate concentration with phosphate buffer. The fixative was gently perfused through the perilymphatic spaces of the cochlea through the hole in Scala tympani and escaped through the oval window. The addition of a small amount of 0~0, to the glutaraldehyde prefixative was following a procedure described by Eisenman and Alfert (1982). Some of the fixatives also contained K,FeCN, or K,CrO, which, based upon our experience, have been shown to improve fixation quality. The fixative was delivered using a micropipet attached to a 1 ml syringe with a 25 gage Butterfly (Abbott) infusion tube. Care was taken to avoid puncture of the round window membrane. Perilymphatic perfusion of the fixative continued for 5 min. The time from decapitation to the introduction of fixative did not exceed 3 min. After perilymphatic perfusion, the round window membrane was opened and the perilymphatic spaces were perfused using a 1 ml syringe fitted with an infusion tube. The co&leas were immersed in the same fixative and rotated for 2 h. After fixation, cochleas were decalcified by immersion in a 15%
155
aqueous solution of disodium EDTA with continuous rotation. Decalcification of the cochleas was complete in 3 days if the cochleas were perfused with fresh EDTA twice daily and continuously rotated. It has been shown previously that decalcification of tissue in EDTA does not interfere with AB staining (Charman and Reid, 1972). After decalcification, cochleas were transferred to a dissecting solution consisting of 0.1 M sodium acetate buffer (pH 5.8) and 0.05-0.25 M MgCl,. AB 8GX was obtained from Sigma and prepared as a 1% stock solution in distilled water. The AB staining solutions were prepared fresh and consisted of 0.1% AB in 0.1 M sodium acetate buffer (pH 5.8) with 0.05-0.25 M MgCl,. The MgCl, was first dissolved in the buffer and then the AB was added to the staining solution immediately prior to staining the cochlea. Cochleas were cut along a mid-modiolar plane using a single-edge razor blade. The cochlear halves were labelled ‘A’ and ‘B’, with the B half containing the extreme basal end or hook. The 7 half-turns of the Scala media were perfused, while immersed in dissecting solution, with a gentle stream of AB using a micropipet. The half-turns were perfused from both cut ends for approximately 10 s. We attempted to perfuse the AB only through the Scala media. After staining each half-turn, the basilar membrane containing the organ of Corti, was dissected free from surrounding cochlear tissues
TABLE
generally following the procedures of Axelsson et al. (1975). This was done by cutting through the optic capsule at each of the 7 half-turns which were labelled: hook, lA, lB, 2A, 2B, 3A, 3B. The otic capsule was gently teased from the spiral ligament using a small curved pick. The spiral ligament, stria vascularis and part of the spiral larnina were removed from the basilar membrane by several small razor blade cuts. Excess tissue was trimmed from the basilar membrane so that it would lie flat on the slide. The tectorial membrane was removed from the organ of Corti and spiral limbus by grasping one edge of it with sharp forceps and peeling it away. The tectorial membrane could usually be removed in 1 or more pieces without damage to the underlying hair cells. The half-turns containing the organ of Corti and pieces of the tectorial membrane and the stria vascularis were directly mounted (from the dissecting solution) into Aqua Mount (Sigma). The half-turns were mounted on the slide with the apical surface of the hair cells nearest the coverslip. Tables I, II and III show the types of fixatives and staining solutions that we used to investigate the characteristics of AB of cochlear tissues. Results Tables 1, II and III summarize cerning AB staining of cochlear
our results contissues. Table I
I
ALDEHYDE
FIXATION
Fixation
Time
N
MgCl,
4% Formalin
24 h
3
0.05 0.10 0.125 0.25 0.05 0.10 0.125 0.25 0.05 0.10 0.125 0.25
2% Glut.
3h
1
2% Glut. + 0.4% K ,CrO,
3h
1
[Ml
NS
ST
TM
SV
++ + + ++
+ ++ + _
++ +++ +++ ++ NA NA NA NA NA NA NA NA
+ + + + _ _ _
+ it +
++ +
N, no. of cochleas; NS. nonspecific; ST, stereocilia; TM, tectorial membrane; SV, stria vascularis: tunnel crossing fibers; NA, not available; -, no staining: + to + + + +, least to greatest staining.
SP
_ _ _
+ + + + + + + + ++ ++ ++ it
SP, spiral
prominence;
_ _
_ _ _ _
TXF
TXF,
156
Fig. 1. A light micrograph cochlear tissues obscured
showing AB staining in 0.1 M MgCl, recognition of the stereocilia. X 1530.
of the organ
shows the effect of aldehyde fixation on AB staining. The formalin fixation group follows the original CEC method of Scott (1960) except that the tissues had been decalcified with EDTA. The hair cell stereocilia, tectorial membrane, tunnel crossing fibers and the stria vascularis were the most intensely stained cochlear tissues. However, staining was unspecific and all cochlear tissue appeared to be moderately stained by AB (Fig. 1). The least diffuse staining of AB was at a MgCl, concentration of 0.1 M. At the highest concentration of
TABLE 0~0,
of Corti after formalin
Diffuse
staming
01 all
MgCl, (0.25 M) the staining was poor due to the fact that the staining solution was unstable and quickly precipitated (within 5 min) after it was prepared. AB staining was better in glutaraldehyde fixed tissue and the tissue also appeared better preserved than tissue fixed with formalin. Table II shows the effect of 0~0, fixation on AB staining. All of the staining solution contained 0.1 M MgCl, since in the aldehyde fixed cochleas we found that this concentration increased staining specificity and was a stable solution. The hair
II
FIXATION
Fixation
Time
N
ST
TM
2% oso, 2% 0~0, 2% 0~0,
2h 2h 2h
3 2 1
+++ + ++
++ + ++
+ 1% K,FeCN, + 0.4% K ,CrO.,
N, no. of co&leas; NS, nonspecific; ST, stereocilia; tunnel
fixation.
crossing
fibers;
NA, not available;
sv _ _
TM, tectorial membrane; SV. stria vascularis; -, no staining; + to + + + + . least to greatest staining.
SP
TXF
_
+
_
_
SP, spiral
prominence;
TXF,
Fig. 2. A light micrograph showing AB staining in 0.1 M M&l1 easily visualized on an unstained background. x 1530.
of the hair cell stereocilia
cell stereocilia were well stained by AB after 0~0, fixation (Fig. 2) whereas, the background staining of other cochlear tissues was much less than after aldehyde fixation alone. The addition of K 3FeCN, TABLE
after 0~0,
fixation.
The stereocilia
are
and K,CrO, to the fixative did not appear to enhance AB staining. Table III shows the effect of prefixation with an aldehyde and postfixation with 0~0,. In half
III
ALDEHYDE
PREFIXATION,
0~0,
POSTFIXATION
Fixation
Time
2% Glut. 0.5% oso, 2% Glut. 0.5% 0~0, + 1% K,FeCN, 2% Glut. 0.5% 0~0, +0.4% K$rO, 2% Glut. + 0.002% 0~0, 2% Glut. 0.5% OSO, 2% Glut. +O.OS% 0~0, 2% Glut. 0.5% oso, 2% Glut. +0.002% 0~0, 2% Glut. 0.5% 0~0, + 1% K,FeCN,
24 h 2h 24 h 2h 24 h 2h 5 min 3h 2h 5 min 3h 2h S min 3h 2h
N
ST
TM
sv
4
i-t
NA
+
1
t- +
++
+
1
i-
+
10
rifi
++
1
+ + +
++
1
+ + +
++
N. no. of cochleas; NS. nonspecific; ST. stereocilia; TM, tectorial membrane; SV, stria vascularis: tunnel crossing fibers; NA, not available; -, no staining: + to + + + t. least to greatest staining.
SP
_
TXF
+
++
_
++
++ SP. spiral
prommence;
TXF.
Fig. 3. The inner and 3 rows of outer hair cells are well stained by AB and show only minimal distortions of their regular, appearance. Background staining of other cochlear tissues is negligible and tissue preservation is good. x 470.
Fig. 4. A light micrograph showing AB staining drug and an intense sound exposure. X 1530.
of giant stereocilia
on the inner hair cells of a chronic
animal
erect
that received a ototoxic
159
of the groups a small amount of 0~0, was combined with the glutaraldehyde prefixative. Stereocilia staining was good in all groups and best in that group which was prefixed in 2% glutaraldehyde containing 0.002% 0~0, (Fig. 3). All four rows of hair cell stereocilia were well stained and showed minimal distortions of the regular, erect appearance of the stereocilia. The tunnel crossing fibers were also stained by AB. The tectorial membrane was also well stained by AB and should be removed from the organ of Corti for good visualization of the hair cell stereocilia. Other cochlear tissues were poorly stained by AB or not stained at all. We also found that AB would stain giant or fused stereocilia present in animals which had received either ototoxic drugs or an intense sound exposure. Fig. 4 shows AB staining of fused and giant stereocilia in a chronic (over 1 year survival) animal that had received ototoxic drugs and an intense sound exposure. Discussion Using the CEC method of Scott (1960) the addition of MgCl, to an acidified, AB staining solution enhanced the specific staining of several cochlear structures. At a concentration of 0.1 M, MgCl, appeared to reduce background staining with AB due to the masking of weakly charged carboxyl anionic sites. Those structures that were strongly stained by AB may represent binding to sulfated polyanions since they have a high CEC. However, tissues fixed only in aldehydes had more background staining and were not as well preserved as tissue fixed only in 0~0,. Since the identification of AGAG using Scott’s method was based upon aldehyde fixation and our best results were with 0~0, fixed tissue we were unable to determine the specific polyanions in cochlear tissues which were responsible for AB staining. AB staining after 0~0, fixation resulted in both enhanced staining of stereocilia and a reduction in background staining. The mechanism of fixation by 0~0, appears to be the reaction with the double bonds present in lipid molecules and the formation of uncharged cyclic esters (Khan et al., 1961). The reduction in the background staining following 0~0, fixation may be due to the elimination of weakly charged anionic sites on the
cell membrane. Since the AGAG lack double bonds, 0~0, does not mask their anionic sites and they remain available for binding with AB. In addition, enhanced AB staining after 0~0, fixation may also be due to its better preservation (compared with aldehydes) of the glycolipids. Furthermore, AB like ruthenium red (Luft, 1971) may form complex molecules with 0~0,; however, AB/OsO, is not nearly as electron dense as ruthenium red/OsO, (Ruggeri et al., 1975). The function of AGAG in the surface membrane of the stereocilia may be to sequester cations from endolymph and thus aid in the transduction process. The same function may also be true for the tectorial membrane since it was intensely stained by AB. Sequestering of cations by AGAG has been demonstrated in cultured heart cells (Langer et al., 1976) and postulated for cochlear stereocilia by Slepecky and Chamberlain (1985). Another presumed function for the negative charge on hair cell stereocilia is to prevent their fusion by electrostatic repulsion (Slepecky and Chamberlain, 1985). If these anionic sites on the AGAG are occupied by cations. then, they may not serve to keep adjacent stereocilia apart by electrostatic repulsion, After AB staining it appears that the AGAG surface coat is quite thick. It is likely that the surface coats of adjacent stereocilia intermingle and that the stereocilia are linked together by AGAG. We have preliminary electron microscopic evidence that this is true using the technique of Behnke and Zelander (1970). Using their method, AB is added to the aldehyde and 0~0, fixatives. This apparently results in the simultaneous stabilization and fixation of the AGAG. The surface coat after this procedure is much thicker than after conventional fixation procedures, Recent physiological evidence (Hudspeth, 1982) indicates that the site of hair cell transduction is at the tips of the stereocilia. We found no specific staining at the stereocilia’s tips. However. the limited resolution of the light microscope may not have permitted us to detect any increased staining along the length of the stereocilia. The absence of AB staining along the endolymphatic surface of the stria vascularis after any of our methods further supports the cation producing role of this tissue. The significance of strong AB staining of
160
the tunnel crossing and spiral bundle nerve fibers is not known but is probably due to the presence of AGAG in the surface membrane of the Schwann cells covering these unmyelinated nerves. This paper has provided evidence for a strong negative charge on the hair ceil stereocilia, tectorial membrane and the tunnel crossing and spiral bundle nerve fibers. We have also provided a reliable method for the recognition of hair cell stereocilia which is compatible with the softsurface preparation method of Axelsson et al. (1975). Specific AB staining of hair cell stereocilia was produced in both normal and abnormal animals. Acknowledgements
The authors would like to thank David C. Muchow for his initial work on AB staining of cochlear hair cell stereocilia. The authors would also like to thank one of the reviewers of this paper for helpful suggestions. This research has been supported by NINCDS grants NS12125 and NS16799. References Axelsson, A., Miller, J. and Larsson. B. (1975): A modified ‘soft surface specimen technique’ for exa~nation of the inner ear. Acta Oto-Laryngol. 80, 362. Behnke. 0. and Zelander, T. (1970): Preservation of intercellular substances by the cationic dye Alcian Blue in preparative procedures for electron microscopy. J. Ultrastruct. Res. 31, 424-438. Behmger, L.F. (1953): Autoradiographic detection of 3% in the membranes of inner ear of rat. Science 118, 520-521. Charman, J. and Reid, L. (1972): The effect of decalcifying fluids on the staining of epithelial mu&s by Alcian Blue. Stain Technol. 47, 173-178. Eisenman, E.A. and Alfert, M. (1982): A new fixation proce-
dure for preserving the ultrastructure of marme invertebrate tissues. J. Microscopy 125. 117-120. Glick, M. and Flowers, H. (1978): Surface membranes. In: The Glycoconjugates, vol. If, Mammalian glycoproteins, glycohpids and proteogiycans, pp. 337-384. Editors: M. Horowitz and W. Pigman. Academic Press, New York. Hudspeth, A.J. (1982): Extracellular current flow and the sate of transduction by vertebrate hair cells. J. Neurosci. 2. l--10. Katagiri, S., Kawamoto, K.. Hori, K. and Watanukr, K. (1968): Some surface views of the inner ear by light microscopy. Acta Qto-laryngol. 66, 493-507. Khan, A., Riemersma, J. and Booij, H. (1961): ‘The reactions of osmium tetroxide with lipids and other compounds. .I. Histochem. Cytochem. 9, 560-563. Langer, G.A., Frank, J.S., Nudd, L.M. and Seraydarian, K. (1976): Sialic acid: effect of its removal on calcium exchangeability of cultured heart cells. Science 193. 1013-1015. Luft, J. (1971): Ruthenium red and violet. 1. Chemistry, purification. methods of use for electron microscopy and mechanism of action. Anat. Rec. 171, 347-368. Ruggeri, A.. Dell’Orbo, C. and Qua&, D. (1975): Electron microscopic visualization of proteoglycans with Afcian Blue. Histochem. J. 7, 187-197. Saito, H. (1982): dtolaryngological studies of glycosaminoglycans and proteoglycans. In: Glycosaminoglycans and proteoglycans in physiological and pathological processes of body systems, pp. 461-479. Editors: R.S. Varma. R. Varma and P.A. Warren. Karger, New York. Scott, J.E. (1960): Aliphatic ammonium salts in the assay of acidic polysaccharides from tissues. Methods B&hem. Anal. 8, 145-197. Scott. J.E. and Dorling, J. (1963): Differential staining of acid glycosaminoglycans (mucopoly~~ha~des) by Alcian Blue in salt solutions. Histochemie 5, 221-233. Scott, J.E., Quintarelli, G. and Dellovo, M.C. (1964): The chemical and histochemical properties of Alcian Blue. I. The mechanism of Alcian Blue staining. Histochemie 4, 73-85. Slepecky, N. and Chamberlain, SC. (1985): The cell coat of inner ear sensory and SUppOrting cells as demonstrated by ruthenium red. Hearing Res. 17, 281-288. Tachibana, M., Saito. H. and Machino, M. (1978): Sulfated acid mucopolysaccharides in the tectorial membrane. Acta Oto-laryngol. 76, 37-46.
153
23 (1986) 153-160
HRR 00792
Alcian Blue staining of cochlear hair cell stereocilia and other cochlear tissues Peter A. Santi * and Craig B. Anderson Department
of Otolaryngolo~,
Unruersity of Minnesota Medical School, Mmneapolw (Received accepted
MN 55455, U.S.A.
9 December 1985; 29 January 1986)
Using whole-mount surface preparations of the chmchilla organ of Corti. the anionic characteristics of cochlear tissues was investigated with the polycationic dye Alcian Blue. Our primary goal was to specifically stain the hair cell stereocilia for the purpose of producing cytocochleograms. To specifically stain hair cell stereocilia we investigated the effects of different fixatives and the ‘critical electrolyte concentration’ [Scott and Dorling (1965) Histochemie 5, 221-2331 of MgCl, on staining specificity. The stereocilia and the tectorial membrane were the most intensely stained cochlear structures and presumably have a strong negative charge. The most important factor in producing specific staining was fixation in osmium tetroxide rather than the addition of an electrolyte to the Alcian Blue staining solution. The use of Alcian Blue produced intense staining of hair cell stereocilia against an unstained background using standard, brightfield light microscopy Alcian
Blue. cochlea,
hair cells, stereocilia
Introduction Alcian Blue 8GX (AB) is a soluble form of copper phthalocyanine which contains up to 4 positively charged isothiouronium groups per molecule (Scott et al., 1964). The mechanism of AB staining has been elucidated by Scott and co-workers (1960, 1964, 1965) and will be briefly reviewed. Due to its cationic nature, AB is strongly held in the negative electrostatic field associated with glycoconjugate polyanions such as chondroitin sulfate, hyaluronic acid, heparin and acid glycosaminoglycans. The majority of the glycoproteins and glycolipids of the cell are associated with the surface membrane and extend into the external environment (Glick and Flowers, 1978). Staining of polyanions is completely blocked by prior treatment with cetyl pyridinium chloride, which is a cationic detergent. The precipitates formed by polyanions with AB are soluble only in strong salt * Present address: S.E.. Minneapolis, 037%5955/86/$03.50
Research East, 2630 MN 55414, U.S.A.
University
Avenue
cb 1986 Elsevier Science Publishers
solutions. The addition of an electrolyte, particularly MgCl 2, to an AB staining solution has been shown to displace the dye from specific binding sites. The basis of this phenomenon is competition between the cations of the electrolyte and the isothiouronium groups on AB for the negative sites on the polyanion. The concentration of salt required to dissolve a given complex depends upon the relative affinities of the competing cation for the polyanion. The ‘critical electrolyte concentration’ (CEC) for several specific negative sites on polyanions has been determined (Scott, 1960; Scott and Dorling, 1965). The anions from the electrolyte added to the staining solution also affect staining intensity. By the blocking of 1 or more of the 4 cationic sites on the AB molecule, more dye is required to combine with the same number of tissue polyanions and the staining appears more intense. The presence of acid glycosaminoglycans (AGAG) in cochlear tissues has been known since the time of Belanger (1953) who reported the presence of sulfated compounds in the tectorial
B.V. (Biomedical
Division)
154
membrane. The AGAG are present on ceil membranes and have a strong negative charge due to the presence of sulfate ester groups and/or carboxyl groups of uranic acids (Scott and Dorling, 1965); thus, they bind cationic dyes. Most of our present knowledge concerning the distribution of AGAG in inner ear tissues is derived from the work of Saito (1982). Using biochemical methods, he found that the distribution of AGAG in cochlear tissues was: 0.1% in the tectorial and Reissner’s membrane, 0.6% in the stria vascularis and spiral ligament and 0.5% in the basilar membrane, spiral limbus and organ of Corti (concentration expressed as: AGAG/dry wt. tissue). However, using histochemical methods involving staining with periodic acid Schiff’s reaction, colloidal iron, methylation and hyaluronidase digestion, neutral red and AB (acidified) Saito (1982) found that AGAG distribution was strongest in the basilar and tectorial membranes and negative in the stria vascularis. Ruthenium red, which is an electron dense, cationic dye also has a strong affinity for the tectorial membrane (Tachibana et al., 1978) and for the hair cell stereocilia (Slepecky and Chamberlain, 1985). The soft-surface preparation method of Axelsson et al. (1975) produces whole-mount preparations of the organ of Corti, which are relatively easy to dissect (compared with plastic embedded whole-mounts), without the loss of significant portions of the basilar membrane. However, these preparations are only lightly and evenly stained by the osmium tetroxide (0~0,) fixative and thus recognition of cochlear cells is usually done using phase or interference microscopy. Since recognition of the hair cell stereocilia is one of the most important criteria in assessing the presence or absence of a hair cell for producing a cytocochleogram, a specific stain for these structures would be desirable. Katagiri et al. (1968) described a method of staining hair cell stereocilia and other cochlear structures using a silver method. However, silver staining methods are known for their capriciousness. In this paper, we describe a method of reliably staining hair cell stereocilia with AB which is compatible with the soft-surface preparation method of Axelsson et al. (1975).
Methods Twenty chinchillas, approximately 1 year old. were used in this study. The animals were anesthetized with an i.p. injection of sodium pentobarbital (60 mg/kg). They were decapitated using bone forceps and scissors. The bullas were separated from the skull by first cutting through the bone between the bullas with scissors. The bullas were then removed by inserting thumbs into the incision and applying upward pressure on the base of the bullas with the index fingers. The bullas were carefully opened to expose the cochleas and the stapes footplate was dislodged from the oval window. A small hole was hand drilled, with a sharpened pick, through the otic capsule of the Scala tympani beneath the first cochlear turn. Several different fixatives were used for this research. Formalin was prepared from paraformaldehyde as a 4% solution in phosphate buffer (pH 7.2). Glutaraldehyde was obtained from Polysciences as a 10% aqueous solution and diluted with phosphate buffer until it was 2% glutaraldehyde in 0.1 M phosphate buffer. 0~0, was also obtained from Polysciences as a 4% aqueous solution and diluted to the appropriate concentration with phosphate buffer. The fixative was gently perfused through the perilymphatic spaces of the cochlea through the hole in Scala tympani and escaped through the oval window. The addition of a small amount of 0~0, to the glutaraldehyde prefixative was following a procedure described by Eisenman and Alfert (1982). Some of the fixatives also contained K,FeCN, or K,CrO, which, based upon our experience, have been shown to improve fixation quality. The fixative was delivered using a micropipet attached to a 1 ml syringe with a 25 gage Butterfly (Abbott) infusion tube. Care was taken to avoid puncture of the round window membrane. Perilymphatic perfusion of the fixative continued for 5 min. The time from decapitation to the introduction of fixative did not exceed 3 min. After perilymphatic perfusion, the round window membrane was opened and the perilymphatic spaces were perfused using a 1 ml syringe fitted with an infusion tube. The co&leas were immersed in the same fixative and rotated for 2 h. After fixation, cochleas were decalcified by immersion in a 15%
155
aqueous solution of disodium EDTA with continuous rotation. Decalcification of the cochleas was complete in 3 days if the cochleas were perfused with fresh EDTA twice daily and continuously rotated. It has been shown previously that decalcification of tissue in EDTA does not interfere with AB staining (Charman and Reid, 1972). After decalcification, cochleas were transferred to a dissecting solution consisting of 0.1 M sodium acetate buffer (pH 5.8) and 0.05-0.25 M MgCl,. AB 8GX was obtained from Sigma and prepared as a 1% stock solution in distilled water. The AB staining solutions were prepared fresh and consisted of 0.1% AB in 0.1 M sodium acetate buffer (pH 5.8) with 0.05-0.25 M MgCl,. The MgCl, was first dissolved in the buffer and then the AB was added to the staining solution immediately prior to staining the cochlea. Cochleas were cut along a mid-modiolar plane using a single-edge razor blade. The cochlear halves were labelled ‘A’ and ‘B’, with the B half containing the extreme basal end or hook. The 7 half-turns of the Scala media were perfused, while immersed in dissecting solution, with a gentle stream of AB using a micropipet. The half-turns were perfused from both cut ends for approximately 10 s. We attempted to perfuse the AB only through the Scala media. After staining each half-turn, the basilar membrane containing the organ of Corti, was dissected free from surrounding cochlear tissues
TABLE
generally following the procedures of Axelsson et al. (1975). This was done by cutting through the optic capsule at each of the 7 half-turns which were labelled: hook, lA, lB, 2A, 2B, 3A, 3B. The otic capsule was gently teased from the spiral ligament using a small curved pick. The spiral ligament, stria vascularis and part of the spiral larnina were removed from the basilar membrane by several small razor blade cuts. Excess tissue was trimmed from the basilar membrane so that it would lie flat on the slide. The tectorial membrane was removed from the organ of Corti and spiral limbus by grasping one edge of it with sharp forceps and peeling it away. The tectorial membrane could usually be removed in 1 or more pieces without damage to the underlying hair cells. The half-turns containing the organ of Corti and pieces of the tectorial membrane and the stria vascularis were directly mounted (from the dissecting solution) into Aqua Mount (Sigma). The half-turns were mounted on the slide with the apical surface of the hair cells nearest the coverslip. Tables I, II and III show the types of fixatives and staining solutions that we used to investigate the characteristics of AB of cochlear tissues. Results Tables 1, II and III summarize cerning AB staining of cochlear
our results contissues. Table I
I
ALDEHYDE
FIXATION
Fixation
Time
N
MgCl,
4% Formalin
24 h
3
0.05 0.10 0.125 0.25 0.05 0.10 0.125 0.25 0.05 0.10 0.125 0.25
2% Glut.
3h
1
2% Glut. + 0.4% K ,CrO,
3h
1
[Ml
NS
ST
TM
SV
++ + + ++
+ ++ + _
++ +++ +++ ++ NA NA NA NA NA NA NA NA
+ + + + _ _ _
+ it +
++ +
N, no. of cochleas; NS. nonspecific; ST, stereocilia; TM, tectorial membrane; SV, stria vascularis: tunnel crossing fibers; NA, not available; -, no staining: + to + + + +, least to greatest staining.
SP
_ _ _
+ + + + + + + + ++ ++ ++ it
SP, spiral
prominence;
_ _
_ _ _ _
TXF
TXF,
156
Fig. 1. A light micrograph cochlear tissues obscured
showing AB staining in 0.1 M MgCl, recognition of the stereocilia. X 1530.
of the organ
shows the effect of aldehyde fixation on AB staining. The formalin fixation group follows the original CEC method of Scott (1960) except that the tissues had been decalcified with EDTA. The hair cell stereocilia, tectorial membrane, tunnel crossing fibers and the stria vascularis were the most intensely stained cochlear tissues. However, staining was unspecific and all cochlear tissue appeared to be moderately stained by AB (Fig. 1). The least diffuse staining of AB was at a MgCl, concentration of 0.1 M. At the highest concentration of
TABLE 0~0,
of Corti after formalin
Diffuse
staming
01 all
MgCl, (0.25 M) the staining was poor due to the fact that the staining solution was unstable and quickly precipitated (within 5 min) after it was prepared. AB staining was better in glutaraldehyde fixed tissue and the tissue also appeared better preserved than tissue fixed with formalin. Table II shows the effect of 0~0, fixation on AB staining. All of the staining solution contained 0.1 M MgCl, since in the aldehyde fixed cochleas we found that this concentration increased staining specificity and was a stable solution. The hair
II
FIXATION
Fixation
Time
N
ST
TM
2% oso, 2% 0~0, 2% 0~0,
2h 2h 2h
3 2 1
+++ + ++
++ + ++
+ 1% K,FeCN, + 0.4% K ,CrO.,
N, no. of co&leas; NS, nonspecific; ST, stereocilia; tunnel
fixation.
crossing
fibers;
NA, not available;
sv _ _
TM, tectorial membrane; SV. stria vascularis; -, no staining; + to + + + + . least to greatest staining.
SP
TXF
_
+
_
_
SP, spiral
prominence;
TXF,
Fig. 2. A light micrograph showing AB staining in 0.1 M M&l1 easily visualized on an unstained background. x 1530.
of the hair cell stereocilia
cell stereocilia were well stained by AB after 0~0, fixation (Fig. 2) whereas, the background staining of other cochlear tissues was much less than after aldehyde fixation alone. The addition of K 3FeCN, TABLE
after 0~0,
fixation.
The stereocilia
are
and K,CrO, to the fixative did not appear to enhance AB staining. Table III shows the effect of prefixation with an aldehyde and postfixation with 0~0,. In half
III
ALDEHYDE
PREFIXATION,
0~0,
POSTFIXATION
Fixation
Time
2% Glut. 0.5% oso, 2% Glut. 0.5% 0~0, + 1% K,FeCN, 2% Glut. 0.5% 0~0, +0.4% K$rO, 2% Glut. + 0.002% 0~0, 2% Glut. 0.5% OSO, 2% Glut. +O.OS% 0~0, 2% Glut. 0.5% oso, 2% Glut. +0.002% 0~0, 2% Glut. 0.5% 0~0, + 1% K,FeCN,
24 h 2h 24 h 2h 24 h 2h 5 min 3h 2h 5 min 3h 2h S min 3h 2h
N
ST
TM
sv
4
i-t
NA
+
1
t- +
++
+
1
i-
+
10
rifi
++
1
+ + +
++
1
+ + +
++
N. no. of cochleas; NS. nonspecific; ST. stereocilia; TM, tectorial membrane; SV, stria vascularis: tunnel crossing fibers; NA, not available; -, no staining: + to + + + t. least to greatest staining.
SP
_
TXF
+
++
_
++
++ SP. spiral
prommence;
TXF.
Fig. 3. The inner and 3 rows of outer hair cells are well stained by AB and show only minimal distortions of their regular, appearance. Background staining of other cochlear tissues is negligible and tissue preservation is good. x 470.
Fig. 4. A light micrograph showing AB staining drug and an intense sound exposure. X 1530.
of giant stereocilia
on the inner hair cells of a chronic
animal
erect
that received a ototoxic
159
of the groups a small amount of 0~0, was combined with the glutaraldehyde prefixative. Stereocilia staining was good in all groups and best in that group which was prefixed in 2% glutaraldehyde containing 0.002% 0~0, (Fig. 3). All four rows of hair cell stereocilia were well stained and showed minimal distortions of the regular, erect appearance of the stereocilia. The tunnel crossing fibers were also stained by AB. The tectorial membrane was also well stained by AB and should be removed from the organ of Corti for good visualization of the hair cell stereocilia. Other cochlear tissues were poorly stained by AB or not stained at all. We also found that AB would stain giant or fused stereocilia present in animals which had received either ototoxic drugs or an intense sound exposure. Fig. 4 shows AB staining of fused and giant stereocilia in a chronic (over 1 year survival) animal that had received ototoxic drugs and an intense sound exposure. Discussion Using the CEC method of Scott (1960) the addition of MgCl, to an acidified, AB staining solution enhanced the specific staining of several cochlear structures. At a concentration of 0.1 M, MgCl, appeared to reduce background staining with AB due to the masking of weakly charged carboxyl anionic sites. Those structures that were strongly stained by AB may represent binding to sulfated polyanions since they have a high CEC. However, tissues fixed only in aldehydes had more background staining and were not as well preserved as tissue fixed only in 0~0,. Since the identification of AGAG using Scott’s method was based upon aldehyde fixation and our best results were with 0~0, fixed tissue we were unable to determine the specific polyanions in cochlear tissues which were responsible for AB staining. AB staining after 0~0, fixation resulted in both enhanced staining of stereocilia and a reduction in background staining. The mechanism of fixation by 0~0, appears to be the reaction with the double bonds present in lipid molecules and the formation of uncharged cyclic esters (Khan et al., 1961). The reduction in the background staining following 0~0, fixation may be due to the elimination of weakly charged anionic sites on the
cell membrane. Since the AGAG lack double bonds, 0~0, does not mask their anionic sites and they remain available for binding with AB. In addition, enhanced AB staining after 0~0, fixation may also be due to its better preservation (compared with aldehydes) of the glycolipids. Furthermore, AB like ruthenium red (Luft, 1971) may form complex molecules with 0~0,; however, AB/OsO, is not nearly as electron dense as ruthenium red/OsO, (Ruggeri et al., 1975). The function of AGAG in the surface membrane of the stereocilia may be to sequester cations from endolymph and thus aid in the transduction process. The same function may also be true for the tectorial membrane since it was intensely stained by AB. Sequestering of cations by AGAG has been demonstrated in cultured heart cells (Langer et al., 1976) and postulated for cochlear stereocilia by Slepecky and Chamberlain (1985). Another presumed function for the negative charge on hair cell stereocilia is to prevent their fusion by electrostatic repulsion (Slepecky and Chamberlain, 1985). If these anionic sites on the AGAG are occupied by cations. then, they may not serve to keep adjacent stereocilia apart by electrostatic repulsion, After AB staining it appears that the AGAG surface coat is quite thick. It is likely that the surface coats of adjacent stereocilia intermingle and that the stereocilia are linked together by AGAG. We have preliminary electron microscopic evidence that this is true using the technique of Behnke and Zelander (1970). Using their method, AB is added to the aldehyde and 0~0, fixatives. This apparently results in the simultaneous stabilization and fixation of the AGAG. The surface coat after this procedure is much thicker than after conventional fixation procedures, Recent physiological evidence (Hudspeth, 1982) indicates that the site of hair cell transduction is at the tips of the stereocilia. We found no specific staining at the stereocilia’s tips. However. the limited resolution of the light microscope may not have permitted us to detect any increased staining along the length of the stereocilia. The absence of AB staining along the endolymphatic surface of the stria vascularis after any of our methods further supports the cation producing role of this tissue. The significance of strong AB staining of
160
the tunnel crossing and spiral bundle nerve fibers is not known but is probably due to the presence of AGAG in the surface membrane of the Schwann cells covering these unmyelinated nerves. This paper has provided evidence for a strong negative charge on the hair ceil stereocilia, tectorial membrane and the tunnel crossing and spiral bundle nerve fibers. We have also provided a reliable method for the recognition of hair cell stereocilia which is compatible with the softsurface preparation method of Axelsson et al. (1975). Specific AB staining of hair cell stereocilia was produced in both normal and abnormal animals. Acknowledgements
The authors would like to thank David C. Muchow for his initial work on AB staining of cochlear hair cell stereocilia. The authors would also like to thank one of the reviewers of this paper for helpful suggestions. This research has been supported by NINCDS grants NS12125 and NS16799. References Axelsson, A., Miller, J. and Larsson. B. (1975): A modified ‘soft surface specimen technique’ for exa~nation of the inner ear. Acta Oto-Laryngol. 80, 362. Behnke. 0. and Zelander, T. (1970): Preservation of intercellular substances by the cationic dye Alcian Blue in preparative procedures for electron microscopy. J. Ultrastruct. Res. 31, 424-438. Behmger, L.F. (1953): Autoradiographic detection of 3% in the membranes of inner ear of rat. Science 118, 520-521. Charman, J. and Reid, L. (1972): The effect of decalcifying fluids on the staining of epithelial mu&s by Alcian Blue. Stain Technol. 47, 173-178. Eisenman, E.A. and Alfert, M. (1982): A new fixation proce-
dure for preserving the ultrastructure of marme invertebrate tissues. J. Microscopy 125. 117-120. Glick, M. and Flowers, H. (1978): Surface membranes. In: The Glycoconjugates, vol. If, Mammalian glycoproteins, glycohpids and proteogiycans, pp. 337-384. Editors: M. Horowitz and W. Pigman. Academic Press, New York. Hudspeth, A.J. (1982): Extracellular current flow and the sate of transduction by vertebrate hair cells. J. Neurosci. 2. l--10. Katagiri, S., Kawamoto, K.. Hori, K. and Watanukr, K. (1968): Some surface views of the inner ear by light microscopy. Acta Qto-laryngol. 66, 493-507. Khan, A., Riemersma, J. and Booij, H. (1961): ‘The reactions of osmium tetroxide with lipids and other compounds. .I. Histochem. Cytochem. 9, 560-563. Langer, G.A., Frank, J.S., Nudd, L.M. and Seraydarian, K. (1976): Sialic acid: effect of its removal on calcium exchangeability of cultured heart cells. Science 193. 1013-1015. Luft, J. (1971): Ruthenium red and violet. 1. Chemistry, purification. methods of use for electron microscopy and mechanism of action. Anat. Rec. 171, 347-368. Ruggeri, A.. Dell’Orbo, C. and Qua&, D. (1975): Electron microscopic visualization of proteoglycans with Afcian Blue. Histochem. J. 7, 187-197. Saito, H. (1982): dtolaryngological studies of glycosaminoglycans and proteoglycans. In: Glycosaminoglycans and proteoglycans in physiological and pathological processes of body systems, pp. 461-479. Editors: R.S. Varma. R. Varma and P.A. Warren. Karger, New York. Scott, J.E. (1960): Aliphatic ammonium salts in the assay of acidic polysaccharides from tissues. Methods B&hem. Anal. 8, 145-197. Scott. J.E. and Dorling, J. (1963): Differential staining of acid glycosaminoglycans (mucopoly~~ha~des) by Alcian Blue in salt solutions. Histochemie 5, 221-233. Scott, J.E., Quintarelli, G. and Dellovo, M.C. (1964): The chemical and histochemical properties of Alcian Blue. I. The mechanism of Alcian Blue staining. Histochemie 4, 73-85. Slepecky, N. and Chamberlain, SC. (1985): The cell coat of inner ear sensory and SUppOrting cells as demonstrated by ruthenium red. Hearing Res. 17, 281-288. Tachibana, M., Saito. H. and Machino, M. (1978): Sulfated acid mucopolysaccharides in the tectorial membrane. Acta Oto-laryngol. 76, 37-46.