ORL 37: 214-222(1975)
Alkaline Phosphatase Activity in the Inner Ear of Fetuses, Newborn and Adult Guinea Pigs Fumihisa Hiraide and Yasuya Nomura Department of Otolaryngology, University of Tokyo, Tokyo
Key Words. Alkaline phosphatase • Developing inner ear • Histochemistry Abstract. The distribution pattern of alkaline phosphatase in the inner ear of fetuses, newborn and adult guinea pigs is described. In the inner ear of fetuses, remarkable alkaline phosphatase was noted throughout the entire tissue of spiral ligament, statoacoustic nerve fibers and the wall of small blood vessels. In newborns, however, less enzyme activity was seen in the spiral ligament and statoacoustic nerve. In adult guinea pigs, no enzyme activity was detectable in the spiral ligament and statoacoustic nerve fibers except for the wall of small blood vessels and efferent nerve fibers. It is evident that the distribution pattern of alkaline phosphatase is altered during the development of the inner ear of the guinea pig.
Histochemical studies on alkaline phosphatase in the inner ear of several mammals have been reported by several authors (7—9, 11 —13, 15). There is disagreement, however, concerning the distribution of this enzyme in the inner ear tissues. There are three important reasons for lack of agreement. First there are differences in applied techniques for demonstrating the enzyme. This was discussed in detail elsewhere (10). The second is due to the fact that alkaline phosphatase shows greater species variability than other enzymes that have been studied histochemically in the various organs (9). The third is that the distribu tion pattern of this enzyme may change during development (1). In the present study, it is attempted to describe the distribution pattern of alkaline phosphatase in the inner ear of fetuses, newborn and adult guinea pigs, using Burstone’s simultaneous coupling azo dye method, and to discuss the findings in relation to pertinent data obtained by other investigators.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Received: October 3, 1974; accepted: December 16, 1974.
Hiraide/Nomura
215
Materials and Method Six fetus guinea pigs (weighing 50 70 g), 8 newborn (0.5-3 days) and 10 adult guinea pigs (weighing approximately 250 g) were used in this study. The animals were decapitated after administration of intraperitoneal Nembutal or ether inhalation. The inner ears were immediately removed and fixed in a 4 % solution of neutral formol calcium for 24 h at 4 °C. Then the tissues were decalcified in a 5 % buffered solution ofEDTA for 4 - 7 days (6). For some fetal temporal bones, the decalcifying process was excluded. The blocks of decalcified or undecalcified inner ears were frozen and mounted for sectioning in a cryostat (-2 0 °C). The sectioned specimens were put on cover glasses, thawed slowly and dried at room temperature for 3 -5 min. Then the specimens were incubated for 2 -1 0 h at 4 °C in a media containing naphthol AS-TR phosphate as a substrate and fast red-violet LB salt as a coupler (5). In most cases, methyl green was used as a nuclear stain. For control specimens, the methods of Barka and Anderson (3) were used to distinguish false positive staining from genuine enzyme reactions. The stained specimens were mounted in glycerin jelly for lightmicroscopic examination.
Results In the cochlea of the fetuses, strong alkaline phosphatase activity was seen in the wall of radiating arterioles and in some capillaries in the upper spiral ligament and throughout the entire tissues of the spiral ligament. The stria
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Fig. 1. Fetus. Strong alkaline phosphatase activity is seen throughout the spiral liga ment (SL) and in the wall of radiating arterioles (arrow) in the upper spiral ligament. OC = Organ of Corti; SV = stria vascularis.
Hiraide/Nomura
216
vascularis did not show any enzyme activity except for some capillaries and bordering tissues to the spiral ligament. The sensory and supporting cells composing the organ of Corti failed to show the obvious enzyme activity. However, remarkable activity was detectable in the nerve endings of sensory hair cells and nerve fibers crossing the tunnel spaces (fig. 1). No reaction products occurred in the tectorial membrane, limbus cells and outer and inner sulcus cells. Reissner’s membrane had little reaction product, especially in the epithelial cells lining the perilymphatic space. Considerable amounts of azo dye granules were observed in the cochlear nerve fibers and on or near the spiral ganglion cells. A much greater amount of reaction products was present in the efferent nerve fibers than the afferent fibers. In the fetal vestibulum, some type of sensory epithelial cells of maculae utriculi and sacculi and cristae ampullares revealed strong enzyme activity. Vestibular nerve fibers and ganglion cells also contained considerable amounts of azo dye granules. The other epithelial cells lining the vestibular spaces remained negative except for the wall of small blood vessels. It is of interest to note that the sensory hairs of vestibular sensory cells were extremely rich in alkaline phosphatase. The enzyme distribution pattern in the cochlea of newborns (0 .5 -3 days after birth) was almost identical with that seen in fetal guinea pigs. However, less
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Fig. 2. Newborn. The alkaline phosphatase activity in cochlear afferent nerve fibers (CAN) is less compared with fetal nerve fibers. However, the enzyme activity of efferent nerve fibers remains intensively positive. IGB = Intraganglionic spiral bundle; PC = plexus cochlearis; SGC = spiral ganglion cells.
Alkaline Phosphatase Activity in the Inner Ear
217
enzyme activity was found in the cochlear and vestibular tissues of newborns than those of fetuses. Alkaline phosphatase activity in afferent statoacoustic nerve fibers was less than that seen in fetal nerve fibers. However, the enzyme activity of efferent nerve fibers remained intensively positive and a strong reaction was also observed in the plexus cochlearis (fig. 2). Weak enzyme activity was still noted in the spiral ligament of newborn animals. In the inner ear of adult guinea pigs, azo dye reaction products were clearly demonstrated in the efferent nerve fibers and the wall of the small blood vessels, such as arterioles and capillaries. Remarkable alkaline phosphatase activity was seen in the nerve endings at the base of sensory hair cells, and also was strongly detectable in the nerve fibers of inner, outer and tunnel spiral bundles. No enzyme activity, however, was found in the sensory hair cells and supporting cells of the organ of Corti and limbus cells, inner and outer sulcus cells (fig. 3). No azo dye granules were found in the cells of stria vascularis and spiral ligament except in the wall of small blood vessels. However, those dye granules were occasionally seen in the tissue between the stria vascularis and spiral liga ment. Marked enzyme activity occurred in radiating arterioles and in some capil laries of the spiral ligament and stria vascularis. However, collecting venules and some capillaries (presumably the distal end of capillaries) had no demonstrable enzyme activity. Reissner’s membrane and tectorial membrane did not show any enzyme
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Fig. 3. Adult. Remarkable alkaline phosphatase activity is seen in the nerve endings at the base of the sensory hair cells, and is also detectable in the nerve fibers crossing the tunnel space. VS = Vas spirale.
Hiraide/Nomura
218
Fig. 4. Adult. Marked alkaline phosphatase is present in the cochlear efferent nerve fibers (arrows). C = Capillary; CAN = cochlear afferent nerve fibers; SGC = spiral ganglion cells.
activity. Small blood vessels in the cochlear and vestibular soft tissues showed moderate to strong enzyme activity. Intense enzyme activity was observed in the intraganglionic spiral bundles and peripheral parts of spiral ganglion cells. Marked enzyme activity was also noted in tire efferent nerve fibers of the modiolus (fig. 4). Strong activity of alkaline phosphatase was present on the sensory hairs and in or near some of the vestibular sensory cells. No histochemical reaction prod ucts appeared in other epithelial cells lining the vestibular spaces. Control speci mens showed no histochemical reaction.
Recently, the application of azo coupling methods has been increasingly employed for demonstrating alkaline phosphatase, because this azo dye method
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Discussion
Alkaline Phosphatase Activity in the Inner Ear
219
F e tu s
N e w b o rn
A d u lt
can avoid the nuclear and background stainings frequently observed with Gomori’s salt-metal technique. These stainings are generally regarded as artifactual products (10). The present investigation has revealed that the alkaline phosphatase distribution pattern in the inner ear of postnatal guinea pigs was altered from that of prenatal animals. It is widely known that embryonic tissues contain a much greater amount of alkaline phosphatase than adult tissues in various animal organs (1). As a matter of fact, the present study has shown that alkaline phosphatase was abundant in the fetal spiral ligament and cochlear and vestibular nerves. However, this enzyme activity disappeared from these tissues within 3 -7 days after birth. The basic chemical role of alkaline phosphatase in development is still unknown. It is likely, however, that among the diverse chemical mechanisms, that lead to organ development, a considerable portion of tissue differentiation may require an invariable factor involving phosphorylation
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Fig. 5. Difference in alkaline phosphatase activity in developing guinea pig cochlea.
220
and dephosphorylation, which necessitates the presence of alkaline phospha tase. Rauch (12) reported that the activity of this enzyme was seen in the capil laries, spiral ligament and periosteal layer of guinea pig fetuses. He also found considerable enzyme activity in areas at the base of sensory hair cells of the organ of Corti and in Reissner’s membrane. His histochemical findings agree with the results of the present observations on the fetal guinea pigs. In respect to alkaline phosphatase in the inner ear of adult guinea pig, there are contradictory reports on the localization. Vinnikov and Titova (15) stated that this enzyme was positive in all the structures of the organ of Corti as well as the stria vascularis. In the present histochemical study, however, no marked activity of alkaline phosphatase was noted in the organ of Corti except for nerve endings and some nerve fibers. Only the endothelial cells of small vessels were positive in the stria vascularis. This difference may be attributed to the tech nique used rather than to the enzyme distributions. The present investigation on the alkaline phosphatase distribution pattern of adult guinea pigs was in substan tial agreement with those recently reported by Schätzte and Westernhagen (13). They demonstrated a high enzyme activity at the base of the sensory hair cells and in the wall of small blood vessels running underneath the organ of Corti by applying both azo dye and salt-metal methods. They also found strong enzyme activity in the cochlear nerve fibers. In the present study, however, no detectable enzyme activity was present in the coch lear nerve fibers of adult guinea pigs except for the efferent nerve fibers. Pre sumably, they might have used newborn guinea pigs for their study, because considerable activity of alkaline phosphatase could be always seen in the statoacoustic nerve fibers of newborn guinea pigs. It has been reported that the endothelium of most proximal capillaries and arterioles displays high alkaline phosphatase activity and that is not demonstrable in the wall of venules and distal end of capillaries of guinea pig inner ears. Differences in the activity of alkaline phosphatase in the cochlear blood vessels may indicate functional differences in the vessels (9). Similar histo chemical findings on the various organs have been reported by several authors (2). Adenosine triphosphatase is a kind of phosphate-liberating enzyme like alka line phosphatase. It has been proposed that alkaline phosphatase and ATPase play the significant part in the phenomenon of permeability, i.e. both enzymes are thought to be concerned with the energy supply in the phosphatidic acid cycle. However, high ATPase activity was always present in the wall of arterioles, capillaries and venules of the inner ear of the adult guinea pig (unpublished). There may be an obvious functional difference between alkaline phosphatase and ATPase in the inner ear blood vessels. The prominent alkaline phosphatase reaction in the cochlear efferent
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Hiraide/Nomura
Alkaline Phosphatase Activity in the Inner Ear
221
nervous system of guinea pig was observed in the present study. Detailed inves tigations on the distribution of this enzyme in the efferent nervous system of this animal have been described elsewhere (7). The consistent appearance of this enzyme in the efferent nervous system and certain small blood vessels of guinea pigs suggests that the possible function of this enzyme may be in the nervous transmission and the transport process. The alkaline phosphatase has been discussed in connection with synaptic trans mission and blood-brain barriers in the central nervous system (4, 14). From the present study it may be concluded that the distribution pattern of alkaline phosphatase is altered during the development of the inner ear of the guinea pig.
Zusammenfassung Die Verteilungsform von alkalischer Phosphatase im Innenohr von fetalen, neuge borenen und erwachsenen Meerschweinchen wird beschrieben. Im Innenohr von Feten wurde im gesamten Gewebe des Ligamentum spirale alkalische Phosphatase in beachtlicher Menge gefunden, vor allem auch in Nervenfasern und Wänden der kleinen Blutgefässe. Bei neugeborenen Meerschweinchen wurde eine geringere Enzymaktivität im Ligamentum spi rale sowie im Nervus cochlearis festgestellt. Keine Enzymaktivität konnte im Ligamentum spirale und den Hörnervenfasern von erwachsenen Meerschweinchen nachgewiesen werden, ausser in den Gefässwänden der kleinen Blutgefässe und in den efferenten Nervenfasern. Es ist offensichtlich, dass sich die Verteilungsform der alkalischen Phosphatase im Innenohr im Laufe der Entwicklung des Meerschweinchens wandelt.
Résumé Le mode de distribution de la phosphatase alcaline dans l’oreille interne de cobayes fœtaux, nouveaux-nés et adultes est décrit. Dans l’oreille interne du fœtus, une quantité remarquable de phosphatase alcaline a été constatée dans l'ensemble des tissus constituant le ligament spiral, les fibres du nerf stato-acoustique de même que dans la paroi des petits vaisseaux sanguins. Chez les nouveaux-nés par contre, une moins grande activité enzyma tique fut trouvée dans le ligament spiral et dans le nerf stato-acoustique. Chez les cobayes adultes, aucune activité enzymatique n’a été décelée dans le ligament spiral ni dans les fibres du nerf stato-acoustique, à l’exception de la paroi des petits vaisseaux sanguins et des fibres nerveuses efférentes. Il est donc évident que le mode de distribution de la phosphatase alcaline change au cours du développement de l’oreille interne du cobaye.
1 2 3
Adams, C.W.M.: Neurohistochemistry, p. 300 (Elsevier, London 1965). Bannister, R.G. and Romanul, F.C.A.: The localization of alkaline phosphatase activity in cerebral blood vessels. J. Neurol. Neurosurg. Psychiat. 26: 333-340 (1963). Barka, T. and Anderson, P.J.: Histochemistry, theory, practice, and bibliography, p. 222 (Harper & Row, New York 1963).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
References
Hiraide/Nomura
5 6 7 8 9 10 11 12 13
14 15
Bourne, G.H.: Histochemical demonstration of phosphatases in the central nervous system of the rat. Expl Cell Res., suppl. 5, p. 101 (1958). Burstone, M.S.: Histochemical comparison of naphthol AS phosphates for the demon stration of phosphatases. J. natn. Cancer Inst. 20: 601-616 (1958). Freiman, D.G.: Organic chelating agent in the demineralization of bone for histo chemical study of alkaline phosphatase. Am. J. clin. Path. 24: 227-235 (1954). Hiraide, F.: Alkaline phosphatase activity in the efferent nervous system of the inner ear. Acta otol. 69: 286-293 (1970). Kirikae, /.; Nomura, Y., and Hiraide, F.: The capillary in the human cochlea. Acta otol. 67: 1-8 (1969). Nomura, Y. and Hiraide, F. : Cochlear blood vessel. A histochemical method of its demonstration. Archs Otolar. 88: 231-237 (1968). Pearse, A.G.E.: Histochemistry, theoretical and applied, p. 384 (1961). Plotz, E. and Perlman, H.B.: A histochemical study of the cochlea. Laryngoscope 65: 291-312 (1955). Rauch, S.: Biochemie des Hôrorgans, p. 290 (Thieme, Stuttgart 1964). Scliatzle, W. und Westernhagen, B.: Vergleichende Untersuchungen zum histochemischen Nachweis alkalischer Phosphatase in der Meerschweinchenschnecke mit verschiedener Mcthodik. Arch. Ohr.- Nas.- KehlkHeilk. 189: 210 -218 (1967). Tewari, H.B. and Bourne, G.H.: Histochemical studies on the distribution of alkaline phosphatases and 5-nucleotidase in the cerebellum of rat. J. Anat. 97: 65-70 (1963). Vinnikov, Y. and Titova, L.K.: Cited in The organ of Corti, its histophysiology and histochemistry, p. 149 (Consultants Bureau, New York 1965).
Fumihisa Hiraide, Department of Otolaryngology, University of Tokyo, Tokyo (Japan)
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4
222
Alkaline Phosphatase Activity in the Inner Ear of Fetuses, Newborn and Adult Guinea Pigs Fumihisa Hiraide and Yasuya Nomura Department of Otolaryngology, University of Tokyo, Tokyo
Key Words. Alkaline phosphatase • Developing inner ear • Histochemistry Abstract. The distribution pattern of alkaline phosphatase in the inner ear of fetuses, newborn and adult guinea pigs is described. In the inner ear of fetuses, remarkable alkaline phosphatase was noted throughout the entire tissue of spiral ligament, statoacoustic nerve fibers and the wall of small blood vessels. In newborns, however, less enzyme activity was seen in the spiral ligament and statoacoustic nerve. In adult guinea pigs, no enzyme activity was detectable in the spiral ligament and statoacoustic nerve fibers except for the wall of small blood vessels and efferent nerve fibers. It is evident that the distribution pattern of alkaline phosphatase is altered during the development of the inner ear of the guinea pig.
Histochemical studies on alkaline phosphatase in the inner ear of several mammals have been reported by several authors (7—9, 11 —13, 15). There is disagreement, however, concerning the distribution of this enzyme in the inner ear tissues. There are three important reasons for lack of agreement. First there are differences in applied techniques for demonstrating the enzyme. This was discussed in detail elsewhere (10). The second is due to the fact that alkaline phosphatase shows greater species variability than other enzymes that have been studied histochemically in the various organs (9). The third is that the distribu tion pattern of this enzyme may change during development (1). In the present study, it is attempted to describe the distribution pattern of alkaline phosphatase in the inner ear of fetuses, newborn and adult guinea pigs, using Burstone’s simultaneous coupling azo dye method, and to discuss the findings in relation to pertinent data obtained by other investigators.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Received: October 3, 1974; accepted: December 16, 1974.
Hiraide/Nomura
215
Materials and Method Six fetus guinea pigs (weighing 50 70 g), 8 newborn (0.5-3 days) and 10 adult guinea pigs (weighing approximately 250 g) were used in this study. The animals were decapitated after administration of intraperitoneal Nembutal or ether inhalation. The inner ears were immediately removed and fixed in a 4 % solution of neutral formol calcium for 24 h at 4 °C. Then the tissues were decalcified in a 5 % buffered solution ofEDTA for 4 - 7 days (6). For some fetal temporal bones, the decalcifying process was excluded. The blocks of decalcified or undecalcified inner ears were frozen and mounted for sectioning in a cryostat (-2 0 °C). The sectioned specimens were put on cover glasses, thawed slowly and dried at room temperature for 3 -5 min. Then the specimens were incubated for 2 -1 0 h at 4 °C in a media containing naphthol AS-TR phosphate as a substrate and fast red-violet LB salt as a coupler (5). In most cases, methyl green was used as a nuclear stain. For control specimens, the methods of Barka and Anderson (3) were used to distinguish false positive staining from genuine enzyme reactions. The stained specimens were mounted in glycerin jelly for lightmicroscopic examination.
Results In the cochlea of the fetuses, strong alkaline phosphatase activity was seen in the wall of radiating arterioles and in some capillaries in the upper spiral ligament and throughout the entire tissues of the spiral ligament. The stria
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Fig. 1. Fetus. Strong alkaline phosphatase activity is seen throughout the spiral liga ment (SL) and in the wall of radiating arterioles (arrow) in the upper spiral ligament. OC = Organ of Corti; SV = stria vascularis.
Hiraide/Nomura
216
vascularis did not show any enzyme activity except for some capillaries and bordering tissues to the spiral ligament. The sensory and supporting cells composing the organ of Corti failed to show the obvious enzyme activity. However, remarkable activity was detectable in the nerve endings of sensory hair cells and nerve fibers crossing the tunnel spaces (fig. 1). No reaction products occurred in the tectorial membrane, limbus cells and outer and inner sulcus cells. Reissner’s membrane had little reaction product, especially in the epithelial cells lining the perilymphatic space. Considerable amounts of azo dye granules were observed in the cochlear nerve fibers and on or near the spiral ganglion cells. A much greater amount of reaction products was present in the efferent nerve fibers than the afferent fibers. In the fetal vestibulum, some type of sensory epithelial cells of maculae utriculi and sacculi and cristae ampullares revealed strong enzyme activity. Vestibular nerve fibers and ganglion cells also contained considerable amounts of azo dye granules. The other epithelial cells lining the vestibular spaces remained negative except for the wall of small blood vessels. It is of interest to note that the sensory hairs of vestibular sensory cells were extremely rich in alkaline phosphatase. The enzyme distribution pattern in the cochlea of newborns (0 .5 -3 days after birth) was almost identical with that seen in fetal guinea pigs. However, less
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Fig. 2. Newborn. The alkaline phosphatase activity in cochlear afferent nerve fibers (CAN) is less compared with fetal nerve fibers. However, the enzyme activity of efferent nerve fibers remains intensively positive. IGB = Intraganglionic spiral bundle; PC = plexus cochlearis; SGC = spiral ganglion cells.
Alkaline Phosphatase Activity in the Inner Ear
217
enzyme activity was found in the cochlear and vestibular tissues of newborns than those of fetuses. Alkaline phosphatase activity in afferent statoacoustic nerve fibers was less than that seen in fetal nerve fibers. However, the enzyme activity of efferent nerve fibers remained intensively positive and a strong reaction was also observed in the plexus cochlearis (fig. 2). Weak enzyme activity was still noted in the spiral ligament of newborn animals. In the inner ear of adult guinea pigs, azo dye reaction products were clearly demonstrated in the efferent nerve fibers and the wall of the small blood vessels, such as arterioles and capillaries. Remarkable alkaline phosphatase activity was seen in the nerve endings at the base of sensory hair cells, and also was strongly detectable in the nerve fibers of inner, outer and tunnel spiral bundles. No enzyme activity, however, was found in the sensory hair cells and supporting cells of the organ of Corti and limbus cells, inner and outer sulcus cells (fig. 3). No azo dye granules were found in the cells of stria vascularis and spiral ligament except in the wall of small blood vessels. However, those dye granules were occasionally seen in the tissue between the stria vascularis and spiral liga ment. Marked enzyme activity occurred in radiating arterioles and in some capil laries of the spiral ligament and stria vascularis. However, collecting venules and some capillaries (presumably the distal end of capillaries) had no demonstrable enzyme activity. Reissner’s membrane and tectorial membrane did not show any enzyme
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Fig. 3. Adult. Remarkable alkaline phosphatase activity is seen in the nerve endings at the base of the sensory hair cells, and is also detectable in the nerve fibers crossing the tunnel space. VS = Vas spirale.
Hiraide/Nomura
218
Fig. 4. Adult. Marked alkaline phosphatase is present in the cochlear efferent nerve fibers (arrows). C = Capillary; CAN = cochlear afferent nerve fibers; SGC = spiral ganglion cells.
activity. Small blood vessels in the cochlear and vestibular soft tissues showed moderate to strong enzyme activity. Intense enzyme activity was observed in the intraganglionic spiral bundles and peripheral parts of spiral ganglion cells. Marked enzyme activity was also noted in tire efferent nerve fibers of the modiolus (fig. 4). Strong activity of alkaline phosphatase was present on the sensory hairs and in or near some of the vestibular sensory cells. No histochemical reaction prod ucts appeared in other epithelial cells lining the vestibular spaces. Control speci mens showed no histochemical reaction.
Recently, the application of azo coupling methods has been increasingly employed for demonstrating alkaline phosphatase, because this azo dye method
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Discussion
Alkaline Phosphatase Activity in the Inner Ear
219
F e tu s
N e w b o rn
A d u lt
can avoid the nuclear and background stainings frequently observed with Gomori’s salt-metal technique. These stainings are generally regarded as artifactual products (10). The present investigation has revealed that the alkaline phosphatase distribution pattern in the inner ear of postnatal guinea pigs was altered from that of prenatal animals. It is widely known that embryonic tissues contain a much greater amount of alkaline phosphatase than adult tissues in various animal organs (1). As a matter of fact, the present study has shown that alkaline phosphatase was abundant in the fetal spiral ligament and cochlear and vestibular nerves. However, this enzyme activity disappeared from these tissues within 3 -7 days after birth. The basic chemical role of alkaline phosphatase in development is still unknown. It is likely, however, that among the diverse chemical mechanisms, that lead to organ development, a considerable portion of tissue differentiation may require an invariable factor involving phosphorylation
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Fig. 5. Difference in alkaline phosphatase activity in developing guinea pig cochlea.
220
and dephosphorylation, which necessitates the presence of alkaline phospha tase. Rauch (12) reported that the activity of this enzyme was seen in the capil laries, spiral ligament and periosteal layer of guinea pig fetuses. He also found considerable enzyme activity in areas at the base of sensory hair cells of the organ of Corti and in Reissner’s membrane. His histochemical findings agree with the results of the present observations on the fetal guinea pigs. In respect to alkaline phosphatase in the inner ear of adult guinea pig, there are contradictory reports on the localization. Vinnikov and Titova (15) stated that this enzyme was positive in all the structures of the organ of Corti as well as the stria vascularis. In the present histochemical study, however, no marked activity of alkaline phosphatase was noted in the organ of Corti except for nerve endings and some nerve fibers. Only the endothelial cells of small vessels were positive in the stria vascularis. This difference may be attributed to the tech nique used rather than to the enzyme distributions. The present investigation on the alkaline phosphatase distribution pattern of adult guinea pigs was in substan tial agreement with those recently reported by Schätzte and Westernhagen (13). They demonstrated a high enzyme activity at the base of the sensory hair cells and in the wall of small blood vessels running underneath the organ of Corti by applying both azo dye and salt-metal methods. They also found strong enzyme activity in the cochlear nerve fibers. In the present study, however, no detectable enzyme activity was present in the coch lear nerve fibers of adult guinea pigs except for the efferent nerve fibers. Pre sumably, they might have used newborn guinea pigs for their study, because considerable activity of alkaline phosphatase could be always seen in the statoacoustic nerve fibers of newborn guinea pigs. It has been reported that the endothelium of most proximal capillaries and arterioles displays high alkaline phosphatase activity and that is not demonstrable in the wall of venules and distal end of capillaries of guinea pig inner ears. Differences in the activity of alkaline phosphatase in the cochlear blood vessels may indicate functional differences in the vessels (9). Similar histo chemical findings on the various organs have been reported by several authors (2). Adenosine triphosphatase is a kind of phosphate-liberating enzyme like alka line phosphatase. It has been proposed that alkaline phosphatase and ATPase play the significant part in the phenomenon of permeability, i.e. both enzymes are thought to be concerned with the energy supply in the phosphatidic acid cycle. However, high ATPase activity was always present in the wall of arterioles, capillaries and venules of the inner ear of the adult guinea pig (unpublished). There may be an obvious functional difference between alkaline phosphatase and ATPase in the inner ear blood vessels. The prominent alkaline phosphatase reaction in the cochlear efferent
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
Hiraide/Nomura
Alkaline Phosphatase Activity in the Inner Ear
221
nervous system of guinea pig was observed in the present study. Detailed inves tigations on the distribution of this enzyme in the efferent nervous system of this animal have been described elsewhere (7). The consistent appearance of this enzyme in the efferent nervous system and certain small blood vessels of guinea pigs suggests that the possible function of this enzyme may be in the nervous transmission and the transport process. The alkaline phosphatase has been discussed in connection with synaptic trans mission and blood-brain barriers in the central nervous system (4, 14). From the present study it may be concluded that the distribution pattern of alkaline phosphatase is altered during the development of the inner ear of the guinea pig.
Zusammenfassung Die Verteilungsform von alkalischer Phosphatase im Innenohr von fetalen, neuge borenen und erwachsenen Meerschweinchen wird beschrieben. Im Innenohr von Feten wurde im gesamten Gewebe des Ligamentum spirale alkalische Phosphatase in beachtlicher Menge gefunden, vor allem auch in Nervenfasern und Wänden der kleinen Blutgefässe. Bei neugeborenen Meerschweinchen wurde eine geringere Enzymaktivität im Ligamentum spi rale sowie im Nervus cochlearis festgestellt. Keine Enzymaktivität konnte im Ligamentum spirale und den Hörnervenfasern von erwachsenen Meerschweinchen nachgewiesen werden, ausser in den Gefässwänden der kleinen Blutgefässe und in den efferenten Nervenfasern. Es ist offensichtlich, dass sich die Verteilungsform der alkalischen Phosphatase im Innenohr im Laufe der Entwicklung des Meerschweinchens wandelt.
Résumé Le mode de distribution de la phosphatase alcaline dans l’oreille interne de cobayes fœtaux, nouveaux-nés et adultes est décrit. Dans l’oreille interne du fœtus, une quantité remarquable de phosphatase alcaline a été constatée dans l'ensemble des tissus constituant le ligament spiral, les fibres du nerf stato-acoustique de même que dans la paroi des petits vaisseaux sanguins. Chez les nouveaux-nés par contre, une moins grande activité enzyma tique fut trouvée dans le ligament spiral et dans le nerf stato-acoustique. Chez les cobayes adultes, aucune activité enzymatique n’a été décelée dans le ligament spiral ni dans les fibres du nerf stato-acoustique, à l’exception de la paroi des petits vaisseaux sanguins et des fibres nerveuses efférentes. Il est donc évident que le mode de distribution de la phosphatase alcaline change au cours du développement de l’oreille interne du cobaye.
1 2 3
Adams, C.W.M.: Neurohistochemistry, p. 300 (Elsevier, London 1965). Bannister, R.G. and Romanul, F.C.A.: The localization of alkaline phosphatase activity in cerebral blood vessels. J. Neurol. Neurosurg. Psychiat. 26: 333-340 (1963). Barka, T. and Anderson, P.J.: Histochemistry, theory, practice, and bibliography, p. 222 (Harper & Row, New York 1963).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 12:33:54 AM
References
Hiraide/Nomura
5 6 7 8 9 10 11 12 13
14 15
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Fumihisa Hiraide, Department of Otolaryngology, University of Tokyo, Tokyo (Japan)
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