Ann 0101 85: 1976
DELINEATION OF COCHLEAR GLYCOGEN BY ELECTRON MICROSCOPY ARNDT
J.
DUVALL
J.
MARGARET
III,
M.D.
HUKEE
MINNEAPOLIS, MINNESOTA
SUMMARY - Two techniques for the demonstration and identification of glycogen by electron microscopy were applied to the cochlear duct tissues of normal guinea pigs and chinchillas. A modified osmium tetroxide fixative (with potassium ferricyanide) has been described by De Bruijn to selectively stain the glycogen particle. Identification of the stained particle was effected by its selective removal from the tissue with amyloglucosidase, an enzyme specific in its degradation of the glycogen molecule. Glycogen particles were noted in several cell types within the cochlear duct, but concentrations were greater in outer hair cells of both species and in the stria vascularis of the chinchilla. The fact that amyloglucosidase completely eliminated these particles from the liver of both species as well as cochlear tissue, led to the conclusion that these particles are indeed glycogen.
Although the glycogen particle occupies a relatively small volume of the animal cell, it is extremely important to the function of some cell types. The glycogen molecule consists of chains of D-glucose units. These units are linked by a-l,4 bonds except at branching points where the linkages are a-l,6 bonds. Within this glycogen structure, glucose is stored in fewer molecules, thus exerting a lower osmotic pressure on the cell than an equivalent number of free glucose molecules.' Glucose is a major source of energy within the cell, and may be metabolized either aerobically or anaerobically. Respiration ( aerobic metabolism) is by far the more efficient system, 1 mole of glucose yielding 36 moles of ATP when completely metabolized. If respiration is interrupted by lack of oxygen to the cell, it is still possible to derive two moles of ATP from one mole of glucose by the anaerobic conversion of glucose to lactate." This process may be important as an energy reserve for cells that require a constant supply of energy, but are
located in areas of little or remote vasculature, such as the cochlear hair cells. The obvious importance of glycogen as a basic storage unit for energy within the cell, and the probable necessity for the organ of Corti to store energy that may be used either aerobically or anaerobically, led to this study of the presence and distribution of glycogen in the cochlear duct. Biochemical evidence is available for both the presence of glycogen and the enzymes necessary for its formation and degradation in the organ of Corti and the stria vascularis." Light microscopy findings showed increased PAS-positive material (glycogen?) in the chinchilla stria during periods of recovery from acoustic trauma.! The locations of this PAS-positive material coincided with those of amorphous osmophilic aggregates found in osmium-fixed strias examined by electron microscopy. In our early work with glycogen, we employed the PAS technique to demonstrate the presence of glycogen under
From the Department of Otolaryngology, University of Minnesota, Minneapolis, Minnesota. This work supported by National Institute of Neurological and Communicative Disorders and Stroke Grant 2-ROI-NS04615-12. 234
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
COCHLEAR GLYCOGEN
the light microscope. A positive PAS reaction is indicative of a compound containing 1:2 glycol groups in its chemical formula. These groups are oxidized by periodic acid to dialdehyde groups which in tum produce colored reaction products when combined with the Schiff reagent." This chemical characteristic is common to a rather vast range of mucosubstances, one of which is glycogen. The positive identification of glycogen is dependent on its removal from the tissue with a glycogen-digesting enzyme such as amylase, resulting in a negative PAS reaction. Amylase is excellent for the removal of glycogen when applied to deparaffinized sections that allow the enzyme penetration." When amylase was used on epon-embedded specimens in our laboratory, enzyme penetration was found to be negligible. Pearse" also reported no penetration of amylase into epoxy-embedded specimens. Efforts to remove the epon from thick sections with a 3% solution of alcoholic sodium hydroxide by the method of Lane and Europa," resulted in a loss of specificity of the PAS stain." Even if amylase was perfused through the perilymphatic spaces before embedding, enzyme penetration into the stria still did not take place. In part because of these difficulties with amylase penetration, another enzyme, amylo-l,6-glucosidase, was adopted for use in the cochlear tissues according to a technique published by Nahorski.? This enzyme, in combination with its ever-present contaminant, oligo-l,4-+ 1,4 glucantransferase.P is more specific for the removal of glycogen because of the ability to break both the a-l,4 and a-l,6 bonds between the glycosidic groups. Amylases (both a and fJ) only break the a-l,4 bonds.P Amyloglucosidase must be used before any osmium fixation in order for its enzymatic action to occur.P Its use at this early stage eliminates the problem of epon penetration. The resolution of the light microscope and the use of the PAS stain allow a
235
very limited picture of glycogen structure and distribution, and a technique was sought for its delineation by electron microscopy. Carmine, as used by Themann'" and Vosteen'" for ultrastructural study of glycogen, causes considerable tissue damage. We found various heavy metal staining techniques'F'" also unsatisfactory. Drochmans'" utilized a negative staining technique (phosphotungstic acid) to demonstrate the three basic forms of glycogen at the ultrastructural level. The alpha form, commonly found in liver tissue, appears as a tightly knit aggregate of smaller particles. These smaller particles also exist outside the aggregate as single units dispersed in the cytoplasm, and are known as beta forms. The gamma form is the smallest of the particles. and is thought to be a substructure of the beta form, as well as the linkage between those beta forms that exist within the alpha Iorm.l''
If both glycogen particles and ribosomes are stained as occurs in routine osmium tetroxide-phosphate buffer fixation with uranyl acetate and lead citrate staining (Fig. IA), it is difficult to differentiate between the two particles because of their similar size. However, De Bruijn-" has recently described a modified osmium tetroxide fixative (with potassium ferricyanide) that stains glycogen but not ribosomes. This method has been combined with the aforementioned enzyme technique of Nahorski" in order to delineate glycogen ultrastructurally in the cochlear duct of the normal chinchilla and guinea pig. METHOnS AND MATERIALS
Normal young adult guinea pigs (200-300 gm) and chinchillas (300-400 gm ) were maintained on a diet of Purina Chin ChoW® and rolled oats for at least three weeks prior to sacrifice. These animals were anesthetized with 60 mg/kgm of sodium pentabarbital given intraperitoneally. The use of sodium pentabarbital does not cause depletion of glycogen content when it is followed by rapid sacrifice and perfusion.t? Ten to fifteen minutes following the administration of anesthetic the animals were sacrificed by decapitation, and within three minutes both cochleae were perfused
Ralston Purina Co., St. Louis, MO
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
236
DUVALL-HUKEE
Fig. 1. A) Normal chinchilla stria marginal cell stained with standard osmium tetroxide-phosphate buffer fixative. Sections stained with uranyl acetate and lead citrate. Note similarities in staining properties and particle size of the ribosomes (R arrow) and the glycogen particles (G). Mitochondria (M), vesicles (V) and endolymph (E). Approximately 58,OOOX. B) Normal chinchilla stria marginal cell stained with the modified osmium tetroxide fixative. Ribosomes are not stained. Glycogen particles both in cluster formation (G) and as single particles (arrow). Approximately 58,OOOX. C) Normal chinchilla stria marginal cell incubated in amyloglucosidase to remove glycogen before postfixation in the modified osmium tetroxide. GC is probably a former glycogen cluster. Approximately 54,500X.
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
COCHLEAR GLYCOGEN with 2% glutaraldehyde buffered with 0.2 M sodium cacodylate at a pH of 7.4. After four hours fixation in this glutaraldehyde solution, the cochleae were rinsed three times in 0.2 M sodium cacodylate buffer, and stored in this buffer at 4 ° C for three to eight weeks, with one buffer change after 24 hours. To facilitate the penetration of amylo-Lflglucosidase into the stria, it was necessary to separate the stria from the spiral ligament. The bone over the lateral cochlear wall was removed with a micropick, and a I-to-2 mm segment of the lateral wall was removed from each turn. The stria and ligament were then separated by teasing a corner of stria away from the ligament with an iris knife, and gently pulling the stria from the ligament with a microtweezer. If the storage time in cacodylate buffer was less than three weeks, the stria adhered tightly to the ligament, making separation difficult. While the stria and ligament of the chinchilla were easily separated after three weeks storage, separation of these tissues in the guinea pig was difficult, regardless of storage or fixation times. The organ of Corti in both species was easily penetrated by the enzyme without removal from the bony capsule. Tissues from the right cochleae were subjected to amylo-Lfi-glucosidase treatment before postfixation with the modified osmium tetroxide, while the tissues of the left cochleae were only postfixed. To remove glycogen from the tissues of the right cochleae, the tissue segments were incubated in a 9 mg/ml solution of amylo-l,6-glucosidase in acetate buffer 9 for ninety minutes at 45° C. Shorter incubation times and weaker concentrations of enzyme were tried, but then glycogen removal was not complete. The incubation temperature was not crucial in glycogen removal from liver tissue or organ of Corti, but had to be maintained at 45° C for successful removal from the stria vascularis. After incubation in the enzyme solution, the specimens were rinsed thoroughly in three changes of cacodylate buffer over a period of thirty minutes. All tissue from the right and left cochleae was then postfixed for two hours at 0° C in a solution of 0.05 M potassium ferricyanide in 1% osmium tetroxide buffered with 0.2 M sodium cacodylate. Liver tissue from both the guinea pig and chinchilla were subjected to the same fixation, staining, and removal techniques as the cochlear tissues. Liver glycogen has been extensively studied, and was used as a control for the reliability of our staining and enzyme removal techniques. After postfixation, all tissues were rinsed again in cacodylate buffer, dehydrated in a series of ethyl alcohols (70% through absolute), cleared in propylene oxide, and embedded in Epon 812. Thin sections were cut
237
on an LKB Ultratome I" and picked up on fornwar-coated grids (75 mesh). These sections were left unstained, and were examined with an electron microscope..... Cochleae fixed in the above modified osmium tetroxide fixative and embedded in epon, were thick sectioned and stained with PAS reagents to demonstrate mucosubstances by light microscopy. Thick sections from other epon-embedded cochleae, fixed in gluteraldehyde-cacodylate buffer (without osmium postfixation) or in standard osmium tetroxide-phosphate buffer, were also stained with PAS. Two PAS techniques were used, the only difference in the two techniques being the temperature at which the Schiff reagent was mixed. Pearsef recommends Schiff mixed in boiling water, while Preecef advocates a Schiff mixed at room temperature. Only the former method was successful in demonstrating PASpositive material in osmium fixed epon-embedded cochleae. RESULTS
When examined by light microscopy, thick sections of epon-embedded guinea pig cochleae fixed with gluteraldehydecacodylate buffer (without osmium postfixation), demonstrated a brilliant PASpositive reaction in the outer hair cells and no reaction in the stria vascularis. The concentration of PAS-positive particles in the guinea pig outer hair cells seemed greater in the upper turns of the cochlea. These findings are the same as those of Falbe-Hansen." In the cochleae fixed with osmium tetroxide-phosphate buffer, a PAS-positive reaction was noted in the outer hair cells of both the guinea pig and chinchilla, and in the stria vascularis of the chinchilla. When cochleae were fixed in the modified osmium tetroxide (with potassium ferricyanide ), the PAS reaction in the outer hair cells was very weak and difficult to substantiate by light microscopy; however, a strong PAS-positive reaction was still definable in the chinchilla stria. Here the difficulty in identifying the PAS reaction in the outer hair cells would seem to be due primarily to the dispersed nature of the glycogen particles, rather than to a masking effect of the modified osmium tetroxide fixative. When examined by electron microscopy, glycogen is present only in the
.. LKB Instruments Inc., Rockville, MD .... Model EMU-3G, RCA Electronic Components, Harrison, NJ
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
238
DUVALL-HUKEE
Fig. 2. A) Normal chinchilla liver stained with modified osmium tetroxide fixative. Note staining of glycogen a-particles, (G) and absence of ribosomes along the endoplasmic reticulum (ER arrow), mitochondrial "dots" (white arrow). Approximately 55,200X. B) Normal chinchilla liver from the same animal as Figure 2A, incubated in amyloglucosldase before postlixation in the modified osmium tetroxide. Note absence of both glycogen particles and ribosomes. Mitochondrial "dots" (arrow). Approximately 55,200X.
beta form in certain cell types of the chinchilla and guinea pig cochlear duct. These beta particles are found in the marginal and intermediate cells of the stria vascularis, the outer and inner hair cells, the pillar cells, and the Deiters' cells of both species. The particles are not attached to or associated with any intracellular membranes, and with the exception of the chinchilla marginal cells, are scattered as single entities throughout the cellular cytoplasm. No glycogen particles were observed within cell nuclei.
In the marginal cells of the normal chinchilla stria, glycogen particles are found in clusters or dispersed as single particles in the cytoplasm (Fig. IB). These clusters are aggregates of glycogen beta forms, but are not as tightly organized as the alpha form found in the chinchilla liver (Fig. 2A). In the marginal cells of the guinea pig, glycogen particles are fewer and more widely dispersed throughout the cell (Fig. 4A), displaying none of the cluster formations evident in the chinchilla marginal cells. In both species, the intermediate
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
COCHLEAR GLYCOGEN
239
Fig. 3. A) Normal chinchilla third outer hair cell from third tum, stained with modified osmium tetroxide fixative. Glycogen B-particles evenly distributed (arrow). Ribosomes not stained. Approximately 57,700X. B) Normal chinchilla second outer hair cell from third tum, incubated in amyloglucosidase before postfixation in modified osmium tetroxide. Note absence of ribosomes and glycogen particles. Approximately 58,200X.
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
240
DUVALL-HUKEE
Fig. 4. A) Normal guinea pig stria marginal cell stained with modified osmium tetroxide fixative. Note glycogen particles (G) and absence of ribosomes. Approximately 60,500X. B) Normal guinea pig stria marginal cell incubated in amyloglucosidase before postfixation in modified osmium tetroxide. Note absence of glycogen particles and ribosomes. Approximately 60,500X.
cells of the stria contain few glycogen particles, and the basal cells contain none. In the outer hair cells of both animals (Figs. 3A and 5A), glycogen particles are evenly distributed throughout the length of the cell, except in the region beneath the nucleus, where the particles are sparse. Within anyone row of outer hair cells, randomly selected adjacent hair cells may have quite different amounts of glycogen particles; and
there is no evidence for a higher concentration of particles in anyone of the three rows. However, preliminary results indicate a greater outer hair cell glycogen content in the upper turns of the cochleae of both species. In the inner hair cells of the chinchilla and guinea pig, glycogen particles are sparse, and are difficult to differentiate from the vast number of vesicles (Fig. 6). The tops of the outer and inner pillar cells of both species show
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
COCHLEAR GLYCOGEN
241
Fig. 5. A) Normal guinea pig second outer hair cell from second turn, stained with modified osmium tetroxide fixative. Note glycogen particles (G). Approximately 57,OOOX. B) Normal guinea pig second outer hair cell from second turn, incubated in amyloglucosidase before postfixation in modified osmium tetroxide. Note absence of glycogen particles. Approximately 61,OOOX.
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
242
DUVALL-HUKEE
Fig. 6. Normal guinea pig inner hair cell from second tum, stained with modified osmium tetroxide fixative. Note absence of ribosomes along the endoplasmic reticulum (ER) and sparse glycogen particles (G) and numerous vesicles (V). Possible artifact (large arrow) of modified osmium staining technique. Approximately 51,700X.
small colonies of particles scattered in the cytoplasm. The liver of the normal chinchilla and guinea pig contains glycogen in an aggregate form, similar to the rat liver alpha form described by Drochmans.l" He measured the beta form within the liver alpha form, and found the beta form to average 37.5 mu in diameter. The liver beta forms of the chinchilla (also measured within the alpha form) averaged 34 mu and those of the guinea pig 20 mu, The beta form present in the cochlear duct averaged 33 mit in the chinchilla and 24 m[1 in
the guinea pig. After incubation in amyloglucosidase to remove glycogen from the tissue, no evidence of glycogen exists in the strial marginal cells of the chinchilla (Fig. Ie). Glycogen removal was not complete without the separation of stria and ligament. In the guinea pig stria, the enzyme removal of glycogen was not always complete unless the ligament was completely severed from the stria. In Figure 4B, a segment of guinea pig stria is shown in which there are no glycogen particles. The stria was cut away from the ligament to allow
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
243
COCHLEAR GLYCOGEN
complete enzyme penetration. Removal of glycogen by amyloglucosidase was complete and consistent in the outer hair cells (Figs. 3B and 5B) and in the liver (Fig. 2B) of both species. Because of the small amounts of glycogen in the intermediate cells, inner hair cells, and pillar cells of both species, the effectiveness of glycogen removal by the enzyme could not be evaluated with any confidence.
contrast of the ribosome seems to be dependent on the UA and Pb staining and is not a property of the modified osmium tetroxide fixative. Amyloglucosidase treatment has no destructive effects on the ribosome and the intact particle may be demonstrated in the tissue by staining sections with UA and Pb. No glycogen particles were visible after this enzyme treatment and staining.
In the chinchilla and guinea pig outer hair cells and in the liver (Fig. 2A), small granules within the mitchondria are also stained by the modified osmium tetroxide fixative. These mitochondrial "dots" are similar in size to the glycogen particles, but the enzyme (amyloglucosidase) would not remove the dots from the tissue (Fig. 2B). The dots may be deposits of calcium or some other divalent cation,22or they may be glycogen particles protected from the enzyme by the mitochondrial membranes.
Within the glycogen clusters of Figure 1, small stained granules appear within and between the beta particles. In the past, precipitate from the lead citrate stain was thought to be responsible for these granules, but in Figure 1B the sections were not stained with lead citrate. A substructure (gamma form) within the beta form has been demonstrated with a negative staining technique.P DISCUSSION
The ultrastructural delineation of glyFollowing the use of the modified cogen in the cochlear tissues necessitates osmium tetroxide fixative, irregularities fulfillment of these three requirements: in the normally cylindrical shape of the outer hair cells were observed. These 1) Cochlear glycogen must be similar are probably artifacts of the fixative. in size and appearance to biochemicallyDegenerative figures (Fig. 6) resem- proven glycogen that has been isolated bling segments of tangled string were from other tissues. also observed in both the cochlear duct 2) The staining technique must be and liver tissues. These figures were specific in its selective delineation of the initially thought to be glycogen residues glycogen particle. left in the tissues after the enzyme treatment, but this possibility was rejected 3) The particle must be removed when the figures were also found in from the tissue by an enzyme specific tissues not subjected to enzyme treat- in its degradation of the glycogen ment. As these figures do not appear molecule. in tissue fixed with standard osmium Though the PAS-positive material in tetroxide-phosphate buffered fixative, they are probably artifacts of the modi- the cochlear tissue was suspected to be fied osmium tetroxide staining tech- glycogen, the proof of its identity was dependent on its removal from the tisnique. sue with an enzyme specific in its action Glutaraldehyde fixed tissues post- on glycogen. Early in this study, the fixed with either the modified osmium enzymes used were a-amylase and I'Jtetroxide or standard osmium tetroxide, amylase. or a combination of the two. demonstrated no ribosomes unless sec- When these enzymes were used, retions were stained with uranyl acetate moval of glycogen from the scala media (UA) and lead citrate (Pb). All the tissues was not complete. The fact that micrographs included in this paper, with amylase would not penetrate the specithe exception of Figure lA, are of tis- men, even before it was embedded in sues not stained with UA and Pb. The epon, leads us to believe that this failure
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
244
DUVALL-HUKEE
to remove glycogen was due at least in part to a limitation of the enzyme. When the properties of amylase are compared to those of amylo-l,6-glucosidase, the limitations of amylase for glycogen removal are evident. Amylase is capable of severing only the a-l,4 bonds of the glycogen molecule, leaving an undigested core of glucose units around the intact a-l,6 bonds. According to Schramm'" these cores are the "limit dextrin" of the enzyme. However, amyloglucosidase will sever both the a-l,4 and a-l,6 bonds of the glycogen, leaving no limit dextrin/" Amylase is very effective in removing glycogen from tissues where it is associated with little tissue protein (such as liver), but in tissues where the glycogen may be bound to cytoplasmic protein (such as muscle), this glycogen removal is more difficult. 18 The nature of the bonding in such a glycogen-protein complex is unknown.l! but the protein portion may be denatured at temperatures above 45° C.2 However, the use of amylase is not recommended at temperatures above 20° C." Amyloglucosidase, on the other hand, will sever both the a-l,4 and a-l,6 bonds in the glycogen molecule even at a temperature of 55° C." The theoretical advantages of amyloglucosidase are apparent and provide the basis for its use in this experiment. Neither amyloglucosidase nor amylase will penetrate the stria from the endolymphatic space or the spiral ligament. This is not surprising, as horseradish peroxidase experiments25,26 indicate significant isolation of the stria from the remainder of the cochlear duct. The form of glycogen (alpha, beta, or gamma) found in different cell types is thought to be related to the primary function of the cell. The alpha form seems to predominate in cell types involved in carbohydrate storage (such as the liver), and this observation is supported by its slower (than beta form) incorporation of a labeled glucose unit. The beta form is present primarily in cell types requiring a continual
energy supply (such as kidney and cardiac muscle), and these cells show a much greater rate of glycogen turnover. 27 The cochlear duct also requires a constant source of high energy, so the presence of glycogen in the beta form is not surprising. Diameters of beta particles measured in the cochlear duct were quite dissimilar from one species to another. For example, our measurements of beta particle diameters averaged 33 mu in the chinchilla and 24 mu in the guinea pig. Drochmans-" has reported the glycogen particle diameter of rat liver beta forms (measured within the alpha form), to average 37.5 mu, The particle size of glycogen (number of glucose subunits) is dependent on its molecular weight,27 which may vary according to the glycogen source, i.e., specie, tissue.'! The distribution of glycogen throughout the guinea pig cochlear duct was similar to that reported by Vosteen.l! with one major exception. He stated that the glycogen particles in the guinea pig stria are concentrated almost exclusively along the surface membranes of the mitochondria. None of the glycogen particles demonstrated by the modified osmium tetroxide fixative had any affinity for the mitochondrial membranes, and were dispersed, unattached, throughout the cytoplasm. Normally, the cell prefers the aerobic metabolism of glucose, because of its higher yield of ATP. When the oxygen supply is depleted or otherwise inadequate, some cell types are capable of anaerobic metabolism of glucose. One index used for the measurement of anaerobic activity is a decrease in the amount of lactic dehydrogenase (LDH), one of the enzymes responsible for the anaerobic conversion of glucose to lactate. 28 The enzyme (LDH) and the substrate (glycogen) for anaeorbic metabolism are both present in the outer hair cell, and may be demonstrated by histochemical techniques." While levels of ATP were sustained in the organ of Corti even through three minutes of
.. Sigma Chemical Co., St. Louis. MO: product Specification
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
COCHLEAR GLYCOGEN
245
induced ischemia, levels of ATP in the stria decreased rapidly." This is an indication that the organ of Corti does indeed use an anaerobic pathway to sustain cellular functions in times of deficient blood supply.
During the stria's period of recovery, an increased amount of PAS-positive material was present.' Histamine and mannitol given intravenously have also produced changes in the glycogen granulation of the chinchilla stria,"
Misrahy''? reported a decrease in oxygen tension of the endolymph after acoustic insult. This tension is dependent on the oxygen supply to the stria and on oxygen consumption by the organ of Corti, and any decrease may indicate an anaerobic environment for the outer hair cells. 2s Following an exposure of 123 dB narrow band noise for 15 minutes, severe but reversible anatomical changes in the stria vascularis of the chinchilla were noted.'
The use of the modified osmium tetroxide technique with the electron microscope increases the possibility of linking established biochemical data to ultrastructural observations in tissues of these induced states. This technique will greatly facilitate the study of glycolysis and glycogenesis in the cochlear duet, and may elucidate the significance of the glycogen granulation changes in pathological states.
REFERENCES 1. Ryman BE, Whelan WJ: New aspects of glycogen metabolism. Adv EnzymoI34:285417, 1972 2. Lehninger AL: Biochemistry. New York, Worth Publishing Incorporated, 1970 3. Matschinsky FM, Thalmann R: Energy metabolism of the cochlear duct. In Paparella MM: Biochemical Mechanisms in Hearing and Deafness. Springfield, Ill, Charles C Thomas, 1970, p 265-288 4. Duvall AI. Ward WD, Lauhala KE: Stria ultrastructure and vessel transport in acoustic trauma. Ann Otol Rhinol Laryngol 83:498-515, 1974
of glycogens. Adv Carbohydr Chern 12:262298, 1957 12. DeBruijn WC: Glycogen, its chemistry and morphologic appearance in the electron microscope. J Ultra Res 42 :29-50, 1973 13. Themann H: Elektronenoptische untersuchungen uber das glykogen in zellstof/wechsel. Stuttgart, Fischer, 1963 14. Vosteen KH: Elektronenmikroskopische untersuchungen uber die verteilung von glykogen im ductus cochlearis bei meerschweinchen. Pract OlolaryngoI26:400-408, 1964
Boston,
15. Revel JP: Electron microscopy of glycogen. J Histochem Cytochem 12:104-114, 1964
6. Preece A: A Manual for Histologic Techniques. Boston, Little, Brown and Company, 1972
16. Thornell LE: Distinction of glycogen and ribosome particles in cow purkinje fibers by enzymatic digestion en bloc and in sections. J Ultra Res 47:153-168,1974
7. Lane BP, Europa DL: Differential staining of ultrathin sections of epon-embedded tissues for light microscopy. J Histochem Cytochern 13:579-582, 1965
17. Churg J, Mautner W, Grishman E: Silver impregnation for electron microscopy. J Biophys Biochem Cyto 4:841, 1958
5. Pearse AGE: Histochemistry. Little, Brown and Company, 1968
9. Nahorski SR, Rogers KJ: An enzymatic fluorornetric micro method for determination of glycogen. Anal Biochem 49:492-497, 1972
18. Drochmans P, Dantan E: Size distributions of liver glycogen particles. In Whelan WJ (ed}: Proceedings of the Fourth Meeting of the Federation of European Biochemical Societies, Control of Glyco~en Metabolism. New York, Academic Press, 1968, p 187-201
10. Brown DH, Illingworth B: The role of oligo-l,4 ~ 1,4 glucantransferase and amylo1,6-glucosidase in the debranching of glycogen. In Whelan WJ, Cameron MP (eds.): Ciba Foundation Symposium on Control of Glycogen Metabolism. Boston, Little, Brown and Company, 1964, p 139-150
19. Wanson [C, Drochmans P: Detection of phosphorylase with the electron microscope. In Whelan WJ (ed): Proceedings of the Fourth Meeting of the Federation of European Biochemical Societies, Control of Glycogen Metabolism. New York, Academic Press 1968 p 169-177 ' ,
11. Manners DJ: The molecular structure
20. Prasannan KG, Subralunanyam K: Com-
8. Duvall AJ: Unpublished data
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
DUV ALL-HUKEE
246
parative study of the methods of determination of cerebral glycogen by quick-freezing technique and by anesthesia. Indian J Exp BioI 10:69-70, 1972 21. Falbe-Hansen J, Thomsen E: Histochemical studies on glycogen in the cochlea of the normal guinea pig. Acta Otolaryngol (Stockh) 56:429-436, 1963 22. Fawcett DW: The Cell, An Atlas of Fine Structure. Philadelphia, W. B. Saunders Company, 1966, p 80 23. Schramm M: Interaction of e-amylase with glycogen and its hydrolysis products. In Whelan WJ, Cameron MP (eds) . Ciba Foundation Symposium on Control of Glycogen Metabolism. Boston, Little, Brown and Company, 1964, p 179 24. Taylor PM, Whelan WJ: Rabbit muscle arnylo-Lfl-glucosidase: properties and evidence of heterogeneity. In Whelan WJ (ed): Prothe Fedceedings of the Fourth Meeting eration of European Biochemica Societies,
0t
Control of Glycogen Metabolism. New York, Academic Press, 1968, p 102 25. Duvall AJ, Quick CA, Sutherland CR: Horseradish peroxidase in the lateral cochlear wall. Arch Otolaryngol 93:304-316, 1971 26. Duvall AJ, Sutherland CR: Cochlear transport of horseradish peroxidase. Arch Otolaryngol 181:705-714, 1972 27. Lumsden RD: Macromolecular structure of glycogen in some cyclophyllidan and trypanorhynch cestodes. J Parasitol 51:501515, 1965 28. Juhn SK: Biochemistry of the Labyrinth. Rochester, Custom Printing Incorporated, 1973 29. Spoendlin HH, Balogh K: Histochemical localization of dehydrogenases in the cochlea of living animals. Laryngoscope 73:1061-1083, 1963 30. Misrahy GA, Hildreth KM, Clark LC, et 01: Measurements of the pH of the endolymph in the cochlea of guinea pigs. Am J Physiol 194:393-395, 1958
REPRINTS: Arndt J. Duvall III, M.D., Box 478, University of Minnesota Hospitals, Minneapolis, MN 55455 ACNNoWLEDGMENT-The authors wish to express their gratitude to Muriel Gavin for her technical assistance in preparation of specimens for electron microscopy.
AMERICAN BOARD OF OTOLARYNGOLOGY The American Board of Otolaryngology will hold its next certifying examination October 23-27, 1976 at the Palmer House, Chicago, Illinois. The deadline date for applying for the 1976 certifying examination of the American Board of Otolaryngology is May 1, 1976.
THE DEAFNESS RESEARCH FOUNDATION The Deafness Research Foundation has awarded over $300,000 for 31 research projects being carried out during 1976 in universities and medical centers throughout the United States and Canada. The announcement was made by John H. Nichols, [r., Chairman of the Board. Founded in 1958 by Mrs. Hobart C. Ramsey, The Deafness Research Foundation is the only national voluntary health organization devoted primarily to furthering otolaryngological research and related objectives. Since its inception it has directed over $5.3 million toward this goal. The Foundation awards grants once a year for a period beginning January 1. Applications are considered for research directed to all aspects of the ear, i.e., investigation of the function, physiology, biochemistry, and genetics, as well as anatomy, pathology and new methods of treatment. Deadline for acceptance of 1977 grant applications is August 15, 1976. For additional information, write Harry Rosenwasser, M.D., Director of Medical Affairs, The Deafness Research Foundation, 366 Madison Avenue, New York, NY 10017.
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
DELINEATION OF COCHLEAR GLYCOGEN BY ELECTRON MICROSCOPY ARNDT
J.
DUVALL
J.
MARGARET
III,
M.D.
HUKEE
MINNEAPOLIS, MINNESOTA
SUMMARY - Two techniques for the demonstration and identification of glycogen by electron microscopy were applied to the cochlear duct tissues of normal guinea pigs and chinchillas. A modified osmium tetroxide fixative (with potassium ferricyanide) has been described by De Bruijn to selectively stain the glycogen particle. Identification of the stained particle was effected by its selective removal from the tissue with amyloglucosidase, an enzyme specific in its degradation of the glycogen molecule. Glycogen particles were noted in several cell types within the cochlear duct, but concentrations were greater in outer hair cells of both species and in the stria vascularis of the chinchilla. The fact that amyloglucosidase completely eliminated these particles from the liver of both species as well as cochlear tissue, led to the conclusion that these particles are indeed glycogen.
Although the glycogen particle occupies a relatively small volume of the animal cell, it is extremely important to the function of some cell types. The glycogen molecule consists of chains of D-glucose units. These units are linked by a-l,4 bonds except at branching points where the linkages are a-l,6 bonds. Within this glycogen structure, glucose is stored in fewer molecules, thus exerting a lower osmotic pressure on the cell than an equivalent number of free glucose molecules.' Glucose is a major source of energy within the cell, and may be metabolized either aerobically or anaerobically. Respiration ( aerobic metabolism) is by far the more efficient system, 1 mole of glucose yielding 36 moles of ATP when completely metabolized. If respiration is interrupted by lack of oxygen to the cell, it is still possible to derive two moles of ATP from one mole of glucose by the anaerobic conversion of glucose to lactate." This process may be important as an energy reserve for cells that require a constant supply of energy, but are
located in areas of little or remote vasculature, such as the cochlear hair cells. The obvious importance of glycogen as a basic storage unit for energy within the cell, and the probable necessity for the organ of Corti to store energy that may be used either aerobically or anaerobically, led to this study of the presence and distribution of glycogen in the cochlear duct. Biochemical evidence is available for both the presence of glycogen and the enzymes necessary for its formation and degradation in the organ of Corti and the stria vascularis." Light microscopy findings showed increased PAS-positive material (glycogen?) in the chinchilla stria during periods of recovery from acoustic trauma.! The locations of this PAS-positive material coincided with those of amorphous osmophilic aggregates found in osmium-fixed strias examined by electron microscopy. In our early work with glycogen, we employed the PAS technique to demonstrate the presence of glycogen under
From the Department of Otolaryngology, University of Minnesota, Minneapolis, Minnesota. This work supported by National Institute of Neurological and Communicative Disorders and Stroke Grant 2-ROI-NS04615-12. 234
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
COCHLEAR GLYCOGEN
the light microscope. A positive PAS reaction is indicative of a compound containing 1:2 glycol groups in its chemical formula. These groups are oxidized by periodic acid to dialdehyde groups which in tum produce colored reaction products when combined with the Schiff reagent." This chemical characteristic is common to a rather vast range of mucosubstances, one of which is glycogen. The positive identification of glycogen is dependent on its removal from the tissue with a glycogen-digesting enzyme such as amylase, resulting in a negative PAS reaction. Amylase is excellent for the removal of glycogen when applied to deparaffinized sections that allow the enzyme penetration." When amylase was used on epon-embedded specimens in our laboratory, enzyme penetration was found to be negligible. Pearse" also reported no penetration of amylase into epoxy-embedded specimens. Efforts to remove the epon from thick sections with a 3% solution of alcoholic sodium hydroxide by the method of Lane and Europa," resulted in a loss of specificity of the PAS stain." Even if amylase was perfused through the perilymphatic spaces before embedding, enzyme penetration into the stria still did not take place. In part because of these difficulties with amylase penetration, another enzyme, amylo-l,6-glucosidase, was adopted for use in the cochlear tissues according to a technique published by Nahorski.? This enzyme, in combination with its ever-present contaminant, oligo-l,4-+ 1,4 glucantransferase.P is more specific for the removal of glycogen because of the ability to break both the a-l,4 and a-l,6 bonds between the glycosidic groups. Amylases (both a and fJ) only break the a-l,4 bonds.P Amyloglucosidase must be used before any osmium fixation in order for its enzymatic action to occur.P Its use at this early stage eliminates the problem of epon penetration. The resolution of the light microscope and the use of the PAS stain allow a
235
very limited picture of glycogen structure and distribution, and a technique was sought for its delineation by electron microscopy. Carmine, as used by Themann'" and Vosteen'" for ultrastructural study of glycogen, causes considerable tissue damage. We found various heavy metal staining techniques'F'" also unsatisfactory. Drochmans'" utilized a negative staining technique (phosphotungstic acid) to demonstrate the three basic forms of glycogen at the ultrastructural level. The alpha form, commonly found in liver tissue, appears as a tightly knit aggregate of smaller particles. These smaller particles also exist outside the aggregate as single units dispersed in the cytoplasm, and are known as beta forms. The gamma form is the smallest of the particles. and is thought to be a substructure of the beta form, as well as the linkage between those beta forms that exist within the alpha Iorm.l''
If both glycogen particles and ribosomes are stained as occurs in routine osmium tetroxide-phosphate buffer fixation with uranyl acetate and lead citrate staining (Fig. IA), it is difficult to differentiate between the two particles because of their similar size. However, De Bruijn-" has recently described a modified osmium tetroxide fixative (with potassium ferricyanide) that stains glycogen but not ribosomes. This method has been combined with the aforementioned enzyme technique of Nahorski" in order to delineate glycogen ultrastructurally in the cochlear duct of the normal chinchilla and guinea pig. METHOnS AND MATERIALS
Normal young adult guinea pigs (200-300 gm) and chinchillas (300-400 gm ) were maintained on a diet of Purina Chin ChoW® and rolled oats for at least three weeks prior to sacrifice. These animals were anesthetized with 60 mg/kgm of sodium pentabarbital given intraperitoneally. The use of sodium pentabarbital does not cause depletion of glycogen content when it is followed by rapid sacrifice and perfusion.t? Ten to fifteen minutes following the administration of anesthetic the animals were sacrificed by decapitation, and within three minutes both cochleae were perfused
Ralston Purina Co., St. Louis, MO
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
236
DUVALL-HUKEE
Fig. 1. A) Normal chinchilla stria marginal cell stained with standard osmium tetroxide-phosphate buffer fixative. Sections stained with uranyl acetate and lead citrate. Note similarities in staining properties and particle size of the ribosomes (R arrow) and the glycogen particles (G). Mitochondria (M), vesicles (V) and endolymph (E). Approximately 58,OOOX. B) Normal chinchilla stria marginal cell stained with the modified osmium tetroxide fixative. Ribosomes are not stained. Glycogen particles both in cluster formation (G) and as single particles (arrow). Approximately 58,OOOX. C) Normal chinchilla stria marginal cell incubated in amyloglucosidase to remove glycogen before postfixation in the modified osmium tetroxide. GC is probably a former glycogen cluster. Approximately 54,500X.
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
COCHLEAR GLYCOGEN with 2% glutaraldehyde buffered with 0.2 M sodium cacodylate at a pH of 7.4. After four hours fixation in this glutaraldehyde solution, the cochleae were rinsed three times in 0.2 M sodium cacodylate buffer, and stored in this buffer at 4 ° C for three to eight weeks, with one buffer change after 24 hours. To facilitate the penetration of amylo-Lflglucosidase into the stria, it was necessary to separate the stria from the spiral ligament. The bone over the lateral cochlear wall was removed with a micropick, and a I-to-2 mm segment of the lateral wall was removed from each turn. The stria and ligament were then separated by teasing a corner of stria away from the ligament with an iris knife, and gently pulling the stria from the ligament with a microtweezer. If the storage time in cacodylate buffer was less than three weeks, the stria adhered tightly to the ligament, making separation difficult. While the stria and ligament of the chinchilla were easily separated after three weeks storage, separation of these tissues in the guinea pig was difficult, regardless of storage or fixation times. The organ of Corti in both species was easily penetrated by the enzyme without removal from the bony capsule. Tissues from the right cochleae were subjected to amylo-Lfi-glucosidase treatment before postfixation with the modified osmium tetroxide, while the tissues of the left cochleae were only postfixed. To remove glycogen from the tissues of the right cochleae, the tissue segments were incubated in a 9 mg/ml solution of amylo-l,6-glucosidase in acetate buffer 9 for ninety minutes at 45° C. Shorter incubation times and weaker concentrations of enzyme were tried, but then glycogen removal was not complete. The incubation temperature was not crucial in glycogen removal from liver tissue or organ of Corti, but had to be maintained at 45° C for successful removal from the stria vascularis. After incubation in the enzyme solution, the specimens were rinsed thoroughly in three changes of cacodylate buffer over a period of thirty minutes. All tissue from the right and left cochleae was then postfixed for two hours at 0° C in a solution of 0.05 M potassium ferricyanide in 1% osmium tetroxide buffered with 0.2 M sodium cacodylate. Liver tissue from both the guinea pig and chinchilla were subjected to the same fixation, staining, and removal techniques as the cochlear tissues. Liver glycogen has been extensively studied, and was used as a control for the reliability of our staining and enzyme removal techniques. After postfixation, all tissues were rinsed again in cacodylate buffer, dehydrated in a series of ethyl alcohols (70% through absolute), cleared in propylene oxide, and embedded in Epon 812. Thin sections were cut
237
on an LKB Ultratome I" and picked up on fornwar-coated grids (75 mesh). These sections were left unstained, and were examined with an electron microscope..... Cochleae fixed in the above modified osmium tetroxide fixative and embedded in epon, were thick sectioned and stained with PAS reagents to demonstrate mucosubstances by light microscopy. Thick sections from other epon-embedded cochleae, fixed in gluteraldehyde-cacodylate buffer (without osmium postfixation) or in standard osmium tetroxide-phosphate buffer, were also stained with PAS. Two PAS techniques were used, the only difference in the two techniques being the temperature at which the Schiff reagent was mixed. Pearsef recommends Schiff mixed in boiling water, while Preecef advocates a Schiff mixed at room temperature. Only the former method was successful in demonstrating PASpositive material in osmium fixed epon-embedded cochleae. RESULTS
When examined by light microscopy, thick sections of epon-embedded guinea pig cochleae fixed with gluteraldehydecacodylate buffer (without osmium postfixation), demonstrated a brilliant PASpositive reaction in the outer hair cells and no reaction in the stria vascularis. The concentration of PAS-positive particles in the guinea pig outer hair cells seemed greater in the upper turns of the cochlea. These findings are the same as those of Falbe-Hansen." In the cochleae fixed with osmium tetroxide-phosphate buffer, a PAS-positive reaction was noted in the outer hair cells of both the guinea pig and chinchilla, and in the stria vascularis of the chinchilla. When cochleae were fixed in the modified osmium tetroxide (with potassium ferricyanide ), the PAS reaction in the outer hair cells was very weak and difficult to substantiate by light microscopy; however, a strong PAS-positive reaction was still definable in the chinchilla stria. Here the difficulty in identifying the PAS reaction in the outer hair cells would seem to be due primarily to the dispersed nature of the glycogen particles, rather than to a masking effect of the modified osmium tetroxide fixative. When examined by electron microscopy, glycogen is present only in the
.. LKB Instruments Inc., Rockville, MD .... Model EMU-3G, RCA Electronic Components, Harrison, NJ
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
238
DUVALL-HUKEE
Fig. 2. A) Normal chinchilla liver stained with modified osmium tetroxide fixative. Note staining of glycogen a-particles, (G) and absence of ribosomes along the endoplasmic reticulum (ER arrow), mitochondrial "dots" (white arrow). Approximately 55,200X. B) Normal chinchilla liver from the same animal as Figure 2A, incubated in amyloglucosldase before postlixation in the modified osmium tetroxide. Note absence of both glycogen particles and ribosomes. Mitochondrial "dots" (arrow). Approximately 55,200X.
beta form in certain cell types of the chinchilla and guinea pig cochlear duct. These beta particles are found in the marginal and intermediate cells of the stria vascularis, the outer and inner hair cells, the pillar cells, and the Deiters' cells of both species. The particles are not attached to or associated with any intracellular membranes, and with the exception of the chinchilla marginal cells, are scattered as single entities throughout the cellular cytoplasm. No glycogen particles were observed within cell nuclei.
In the marginal cells of the normal chinchilla stria, glycogen particles are found in clusters or dispersed as single particles in the cytoplasm (Fig. IB). These clusters are aggregates of glycogen beta forms, but are not as tightly organized as the alpha form found in the chinchilla liver (Fig. 2A). In the marginal cells of the guinea pig, glycogen particles are fewer and more widely dispersed throughout the cell (Fig. 4A), displaying none of the cluster formations evident in the chinchilla marginal cells. In both species, the intermediate
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
COCHLEAR GLYCOGEN
239
Fig. 3. A) Normal chinchilla third outer hair cell from third tum, stained with modified osmium tetroxide fixative. Glycogen B-particles evenly distributed (arrow). Ribosomes not stained. Approximately 57,700X. B) Normal chinchilla second outer hair cell from third tum, incubated in amyloglucosidase before postfixation in modified osmium tetroxide. Note absence of ribosomes and glycogen particles. Approximately 58,200X.
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
240
DUVALL-HUKEE
Fig. 4. A) Normal guinea pig stria marginal cell stained with modified osmium tetroxide fixative. Note glycogen particles (G) and absence of ribosomes. Approximately 60,500X. B) Normal guinea pig stria marginal cell incubated in amyloglucosidase before postfixation in modified osmium tetroxide. Note absence of glycogen particles and ribosomes. Approximately 60,500X.
cells of the stria contain few glycogen particles, and the basal cells contain none. In the outer hair cells of both animals (Figs. 3A and 5A), glycogen particles are evenly distributed throughout the length of the cell, except in the region beneath the nucleus, where the particles are sparse. Within anyone row of outer hair cells, randomly selected adjacent hair cells may have quite different amounts of glycogen particles; and
there is no evidence for a higher concentration of particles in anyone of the three rows. However, preliminary results indicate a greater outer hair cell glycogen content in the upper turns of the cochleae of both species. In the inner hair cells of the chinchilla and guinea pig, glycogen particles are sparse, and are difficult to differentiate from the vast number of vesicles (Fig. 6). The tops of the outer and inner pillar cells of both species show
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
COCHLEAR GLYCOGEN
241
Fig. 5. A) Normal guinea pig second outer hair cell from second turn, stained with modified osmium tetroxide fixative. Note glycogen particles (G). Approximately 57,OOOX. B) Normal guinea pig second outer hair cell from second turn, incubated in amyloglucosidase before postfixation in modified osmium tetroxide. Note absence of glycogen particles. Approximately 61,OOOX.
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
242
DUVALL-HUKEE
Fig. 6. Normal guinea pig inner hair cell from second tum, stained with modified osmium tetroxide fixative. Note absence of ribosomes along the endoplasmic reticulum (ER) and sparse glycogen particles (G) and numerous vesicles (V). Possible artifact (large arrow) of modified osmium staining technique. Approximately 51,700X.
small colonies of particles scattered in the cytoplasm. The liver of the normal chinchilla and guinea pig contains glycogen in an aggregate form, similar to the rat liver alpha form described by Drochmans.l" He measured the beta form within the liver alpha form, and found the beta form to average 37.5 mu in diameter. The liver beta forms of the chinchilla (also measured within the alpha form) averaged 34 mu and those of the guinea pig 20 mu, The beta form present in the cochlear duct averaged 33 mit in the chinchilla and 24 m[1 in
the guinea pig. After incubation in amyloglucosidase to remove glycogen from the tissue, no evidence of glycogen exists in the strial marginal cells of the chinchilla (Fig. Ie). Glycogen removal was not complete without the separation of stria and ligament. In the guinea pig stria, the enzyme removal of glycogen was not always complete unless the ligament was completely severed from the stria. In Figure 4B, a segment of guinea pig stria is shown in which there are no glycogen particles. The stria was cut away from the ligament to allow
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
243
COCHLEAR GLYCOGEN
complete enzyme penetration. Removal of glycogen by amyloglucosidase was complete and consistent in the outer hair cells (Figs. 3B and 5B) and in the liver (Fig. 2B) of both species. Because of the small amounts of glycogen in the intermediate cells, inner hair cells, and pillar cells of both species, the effectiveness of glycogen removal by the enzyme could not be evaluated with any confidence.
contrast of the ribosome seems to be dependent on the UA and Pb staining and is not a property of the modified osmium tetroxide fixative. Amyloglucosidase treatment has no destructive effects on the ribosome and the intact particle may be demonstrated in the tissue by staining sections with UA and Pb. No glycogen particles were visible after this enzyme treatment and staining.
In the chinchilla and guinea pig outer hair cells and in the liver (Fig. 2A), small granules within the mitchondria are also stained by the modified osmium tetroxide fixative. These mitochondrial "dots" are similar in size to the glycogen particles, but the enzyme (amyloglucosidase) would not remove the dots from the tissue (Fig. 2B). The dots may be deposits of calcium or some other divalent cation,22or they may be glycogen particles protected from the enzyme by the mitochondrial membranes.
Within the glycogen clusters of Figure 1, small stained granules appear within and between the beta particles. In the past, precipitate from the lead citrate stain was thought to be responsible for these granules, but in Figure 1B the sections were not stained with lead citrate. A substructure (gamma form) within the beta form has been demonstrated with a negative staining technique.P DISCUSSION
The ultrastructural delineation of glyFollowing the use of the modified cogen in the cochlear tissues necessitates osmium tetroxide fixative, irregularities fulfillment of these three requirements: in the normally cylindrical shape of the outer hair cells were observed. These 1) Cochlear glycogen must be similar are probably artifacts of the fixative. in size and appearance to biochemicallyDegenerative figures (Fig. 6) resem- proven glycogen that has been isolated bling segments of tangled string were from other tissues. also observed in both the cochlear duct 2) The staining technique must be and liver tissues. These figures were specific in its selective delineation of the initially thought to be glycogen residues glycogen particle. left in the tissues after the enzyme treatment, but this possibility was rejected 3) The particle must be removed when the figures were also found in from the tissue by an enzyme specific tissues not subjected to enzyme treat- in its degradation of the glycogen ment. As these figures do not appear molecule. in tissue fixed with standard osmium Though the PAS-positive material in tetroxide-phosphate buffered fixative, they are probably artifacts of the modi- the cochlear tissue was suspected to be fied osmium tetroxide staining tech- glycogen, the proof of its identity was dependent on its removal from the tisnique. sue with an enzyme specific in its action Glutaraldehyde fixed tissues post- on glycogen. Early in this study, the fixed with either the modified osmium enzymes used were a-amylase and I'Jtetroxide or standard osmium tetroxide, amylase. or a combination of the two. demonstrated no ribosomes unless sec- When these enzymes were used, retions were stained with uranyl acetate moval of glycogen from the scala media (UA) and lead citrate (Pb). All the tissues was not complete. The fact that micrographs included in this paper, with amylase would not penetrate the specithe exception of Figure lA, are of tis- men, even before it was embedded in sues not stained with UA and Pb. The epon, leads us to believe that this failure
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
244
DUVALL-HUKEE
to remove glycogen was due at least in part to a limitation of the enzyme. When the properties of amylase are compared to those of amylo-l,6-glucosidase, the limitations of amylase for glycogen removal are evident. Amylase is capable of severing only the a-l,4 bonds of the glycogen molecule, leaving an undigested core of glucose units around the intact a-l,6 bonds. According to Schramm'" these cores are the "limit dextrin" of the enzyme. However, amyloglucosidase will sever both the a-l,4 and a-l,6 bonds of the glycogen, leaving no limit dextrin/" Amylase is very effective in removing glycogen from tissues where it is associated with little tissue protein (such as liver), but in tissues where the glycogen may be bound to cytoplasmic protein (such as muscle), this glycogen removal is more difficult. 18 The nature of the bonding in such a glycogen-protein complex is unknown.l! but the protein portion may be denatured at temperatures above 45° C.2 However, the use of amylase is not recommended at temperatures above 20° C." Amyloglucosidase, on the other hand, will sever both the a-l,4 and a-l,6 bonds in the glycogen molecule even at a temperature of 55° C." The theoretical advantages of amyloglucosidase are apparent and provide the basis for its use in this experiment. Neither amyloglucosidase nor amylase will penetrate the stria from the endolymphatic space or the spiral ligament. This is not surprising, as horseradish peroxidase experiments25,26 indicate significant isolation of the stria from the remainder of the cochlear duct. The form of glycogen (alpha, beta, or gamma) found in different cell types is thought to be related to the primary function of the cell. The alpha form seems to predominate in cell types involved in carbohydrate storage (such as the liver), and this observation is supported by its slower (than beta form) incorporation of a labeled glucose unit. The beta form is present primarily in cell types requiring a continual
energy supply (such as kidney and cardiac muscle), and these cells show a much greater rate of glycogen turnover. 27 The cochlear duct also requires a constant source of high energy, so the presence of glycogen in the beta form is not surprising. Diameters of beta particles measured in the cochlear duct were quite dissimilar from one species to another. For example, our measurements of beta particle diameters averaged 33 mu in the chinchilla and 24 mu in the guinea pig. Drochmans-" has reported the glycogen particle diameter of rat liver beta forms (measured within the alpha form), to average 37.5 mu, The particle size of glycogen (number of glucose subunits) is dependent on its molecular weight,27 which may vary according to the glycogen source, i.e., specie, tissue.'! The distribution of glycogen throughout the guinea pig cochlear duct was similar to that reported by Vosteen.l! with one major exception. He stated that the glycogen particles in the guinea pig stria are concentrated almost exclusively along the surface membranes of the mitochondria. None of the glycogen particles demonstrated by the modified osmium tetroxide fixative had any affinity for the mitochondrial membranes, and were dispersed, unattached, throughout the cytoplasm. Normally, the cell prefers the aerobic metabolism of glucose, because of its higher yield of ATP. When the oxygen supply is depleted or otherwise inadequate, some cell types are capable of anaerobic metabolism of glucose. One index used for the measurement of anaerobic activity is a decrease in the amount of lactic dehydrogenase (LDH), one of the enzymes responsible for the anaerobic conversion of glucose to lactate. 28 The enzyme (LDH) and the substrate (glycogen) for anaeorbic metabolism are both present in the outer hair cell, and may be demonstrated by histochemical techniques." While levels of ATP were sustained in the organ of Corti even through three minutes of
.. Sigma Chemical Co., St. Louis. MO: product Specification
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
COCHLEAR GLYCOGEN
245
induced ischemia, levels of ATP in the stria decreased rapidly." This is an indication that the organ of Corti does indeed use an anaerobic pathway to sustain cellular functions in times of deficient blood supply.
During the stria's period of recovery, an increased amount of PAS-positive material was present.' Histamine and mannitol given intravenously have also produced changes in the glycogen granulation of the chinchilla stria,"
Misrahy''? reported a decrease in oxygen tension of the endolymph after acoustic insult. This tension is dependent on the oxygen supply to the stria and on oxygen consumption by the organ of Corti, and any decrease may indicate an anaerobic environment for the outer hair cells. 2s Following an exposure of 123 dB narrow band noise for 15 minutes, severe but reversible anatomical changes in the stria vascularis of the chinchilla were noted.'
The use of the modified osmium tetroxide technique with the electron microscope increases the possibility of linking established biochemical data to ultrastructural observations in tissues of these induced states. This technique will greatly facilitate the study of glycolysis and glycogenesis in the cochlear duet, and may elucidate the significance of the glycogen granulation changes in pathological states.
REFERENCES 1. Ryman BE, Whelan WJ: New aspects of glycogen metabolism. Adv EnzymoI34:285417, 1972 2. Lehninger AL: Biochemistry. New York, Worth Publishing Incorporated, 1970 3. Matschinsky FM, Thalmann R: Energy metabolism of the cochlear duct. In Paparella MM: Biochemical Mechanisms in Hearing and Deafness. Springfield, Ill, Charles C Thomas, 1970, p 265-288 4. Duvall AI. Ward WD, Lauhala KE: Stria ultrastructure and vessel transport in acoustic trauma. Ann Otol Rhinol Laryngol 83:498-515, 1974
of glycogens. Adv Carbohydr Chern 12:262298, 1957 12. DeBruijn WC: Glycogen, its chemistry and morphologic appearance in the electron microscope. J Ultra Res 42 :29-50, 1973 13. Themann H: Elektronenoptische untersuchungen uber das glykogen in zellstof/wechsel. Stuttgart, Fischer, 1963 14. Vosteen KH: Elektronenmikroskopische untersuchungen uber die verteilung von glykogen im ductus cochlearis bei meerschweinchen. Pract OlolaryngoI26:400-408, 1964
Boston,
15. Revel JP: Electron microscopy of glycogen. J Histochem Cytochem 12:104-114, 1964
6. Preece A: A Manual for Histologic Techniques. Boston, Little, Brown and Company, 1972
16. Thornell LE: Distinction of glycogen and ribosome particles in cow purkinje fibers by enzymatic digestion en bloc and in sections. J Ultra Res 47:153-168,1974
7. Lane BP, Europa DL: Differential staining of ultrathin sections of epon-embedded tissues for light microscopy. J Histochem Cytochern 13:579-582, 1965
17. Churg J, Mautner W, Grishman E: Silver impregnation for electron microscopy. J Biophys Biochem Cyto 4:841, 1958
5. Pearse AGE: Histochemistry. Little, Brown and Company, 1968
9. Nahorski SR, Rogers KJ: An enzymatic fluorornetric micro method for determination of glycogen. Anal Biochem 49:492-497, 1972
18. Drochmans P, Dantan E: Size distributions of liver glycogen particles. In Whelan WJ (ed}: Proceedings of the Fourth Meeting of the Federation of European Biochemical Societies, Control of Glyco~en Metabolism. New York, Academic Press, 1968, p 187-201
10. Brown DH, Illingworth B: The role of oligo-l,4 ~ 1,4 glucantransferase and amylo1,6-glucosidase in the debranching of glycogen. In Whelan WJ, Cameron MP (eds.): Ciba Foundation Symposium on Control of Glycogen Metabolism. Boston, Little, Brown and Company, 1964, p 139-150
19. Wanson [C, Drochmans P: Detection of phosphorylase with the electron microscope. In Whelan WJ (ed): Proceedings of the Fourth Meeting of the Federation of European Biochemical Societies, Control of Glycogen Metabolism. New York, Academic Press 1968 p 169-177 ' ,
11. Manners DJ: The molecular structure
20. Prasannan KG, Subralunanyam K: Com-
8. Duvall AJ: Unpublished data
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
DUV ALL-HUKEE
246
parative study of the methods of determination of cerebral glycogen by quick-freezing technique and by anesthesia. Indian J Exp BioI 10:69-70, 1972 21. Falbe-Hansen J, Thomsen E: Histochemical studies on glycogen in the cochlea of the normal guinea pig. Acta Otolaryngol (Stockh) 56:429-436, 1963 22. Fawcett DW: The Cell, An Atlas of Fine Structure. Philadelphia, W. B. Saunders Company, 1966, p 80 23. Schramm M: Interaction of e-amylase with glycogen and its hydrolysis products. In Whelan WJ, Cameron MP (eds) . Ciba Foundation Symposium on Control of Glycogen Metabolism. Boston, Little, Brown and Company, 1964, p 179 24. Taylor PM, Whelan WJ: Rabbit muscle arnylo-Lfl-glucosidase: properties and evidence of heterogeneity. In Whelan WJ (ed): Prothe Fedceedings of the Fourth Meeting eration of European Biochemica Societies,
0t
Control of Glycogen Metabolism. New York, Academic Press, 1968, p 102 25. Duvall AJ, Quick CA, Sutherland CR: Horseradish peroxidase in the lateral cochlear wall. Arch Otolaryngol 93:304-316, 1971 26. Duvall AJ, Sutherland CR: Cochlear transport of horseradish peroxidase. Arch Otolaryngol 181:705-714, 1972 27. Lumsden RD: Macromolecular structure of glycogen in some cyclophyllidan and trypanorhynch cestodes. J Parasitol 51:501515, 1965 28. Juhn SK: Biochemistry of the Labyrinth. Rochester, Custom Printing Incorporated, 1973 29. Spoendlin HH, Balogh K: Histochemical localization of dehydrogenases in the cochlea of living animals. Laryngoscope 73:1061-1083, 1963 30. Misrahy GA, Hildreth KM, Clark LC, et 01: Measurements of the pH of the endolymph in the cochlea of guinea pigs. Am J Physiol 194:393-395, 1958
REPRINTS: Arndt J. Duvall III, M.D., Box 478, University of Minnesota Hospitals, Minneapolis, MN 55455 ACNNoWLEDGMENT-The authors wish to express their gratitude to Muriel Gavin for her technical assistance in preparation of specimens for electron microscopy.
AMERICAN BOARD OF OTOLARYNGOLOGY The American Board of Otolaryngology will hold its next certifying examination October 23-27, 1976 at the Palmer House, Chicago, Illinois. The deadline date for applying for the 1976 certifying examination of the American Board of Otolaryngology is May 1, 1976.
THE DEAFNESS RESEARCH FOUNDATION The Deafness Research Foundation has awarded over $300,000 for 31 research projects being carried out during 1976 in universities and medical centers throughout the United States and Canada. The announcement was made by John H. Nichols, [r., Chairman of the Board. Founded in 1958 by Mrs. Hobart C. Ramsey, The Deafness Research Foundation is the only national voluntary health organization devoted primarily to furthering otolaryngological research and related objectives. Since its inception it has directed over $5.3 million toward this goal. The Foundation awards grants once a year for a period beginning January 1. Applications are considered for research directed to all aspects of the ear, i.e., investigation of the function, physiology, biochemistry, and genetics, as well as anatomy, pathology and new methods of treatment. Deadline for acceptance of 1977 grant applications is August 15, 1976. For additional information, write Harry Rosenwasser, M.D., Director of Medical Affairs, The Deafness Research Foundation, 366 Madison Avenue, New York, NY 10017.
Downloaded from aor.sagepub.com at Bobst Library, New York University on June 1, 2015
