Histochemical observation on phosphatase activities of degenerating and regenerating taste buds.код для вставкиСкачать
Histochemical Observation on Phosphatase Activities of Degenerating and Regenerating Taste Buds TAKASHI IWAYAMA AND OSAMI NADA Department of Anatomy, Faculty of Medicine, Kyushu University, Fukuoka, Japan ABSTRACT Using rat's circumvallate papillae, ATPase, alk. Pase and acid Pase of taste buds were observed after the transection of the glossopharyngeal nerve. The taste buds began to disappear after the nerve war cut and were completely lost after ten days. Following the regeneration of the glossopharyngeal nerve, taste buds reappeared from the bottom of the gutters of circumvallate papillae about 25 days after the operation. ATPase was strongly present on the cell membrane of taste bud cells as far as they existed during degeneration and regeneration. Alk. Pase, which is normally localized on the superficial layers of the epithelium overlying the gutters of circumvallate papilla, gradually diminished as the taste buds degenerated and reappeared as the taste buds regenerated; that is, the activity began to diminish three days after the operation, became feeble after ten days and reappeared after 25 days. It is concluded that taste bud cells secrete alk. Pase in the gutters of circumvallate papillae. Acid Pase activity, usually found i n the supranuclear portion of taste bud cells, was intensely reactive during degeneration but did not reappear a t the early stage of regeneration of taste bud cells. Histochemical observations have reIt is of particular interest to examine vealed that three kinds of phosphatase the changes of phosphatase activities after activities are localized in the gustatory re- the operaion on the nerve to a taste bud. gions of the tongue (Iwayama and Nada, The present work, a short account of which '67a,b). Adenosine triphosphatase (ATPase) has already been reported (Iwayama et al., and acid phosphatase (acid Pase) are found '67), was mainly undertaken to clarify to be specific in taste bud cells, while alka- the relation of alk. Pase to taste bud cells. line phosphatase (alk. Pase) is present on MATERIAL AND METHODS the superficial layers of the epithelium The circumvallate papilla of albino rat lining the gutters of circumvallate and foliate papillae. In these papillae, alk. Pase was chosen in this experiment because this activity, which is confined only to the papilla is solely innervated by the glossogutters with taste buds, is probably not pro- pharyngeal nerve, while the foliate papilla duced by such inactive cells as cornified of rat is supplied mostly by this nerve and squamous epithelia but seems to have close partly by the chorda tympani (Kitamura, relationship to taste bud cells. This en- '65). Under anesthesia with an intramuscular zyme, however, is not present on the superficial epithelium of the fungiform papilla administr,ition of pentobarbital sodium where a taste bud is located thereon. Judg- (200 mg/ kg), the glossopharyngeal nerve ing from this point of view, the direct was exposed by a median incision on the correlation of alk. Pase to taste buds is un- neck and separation of the stylohyoid and certain. On the other hand, a number of digastric muscles. The nerve was bilaterexperiments showed that taste buds dis- ally transected at the point after it passes appear and reappear following the dener- over the greater horn of the hyoid bone and vation and regeneration of the innervating just before it enters the tongue muscles. nerve (Meyer, 1897; Olmsted, '20, '21, '22; In the deiiervation experiments, the proxof the glossopharyngeal nerve May, '25; Whiteside, '26; Torrey, '34, '40; imal stump Guth, '57, '58). Received Aug. 13, '68. Accepted Oct. 10, '68. ANAT. REC., 163: 31-38. 31 32 TAKASHI I W A Y A M A A N D O S A M I N A D A Four to seven days after the operation, taste buds, as well as taste bud cells, apparently diminished in number. ATPase still persisted on the membrane of taste bud cells although it might be less reactive (fig. 6 ) . Acid Pase was intensely present in the cytoplasm of a few surviving cells (fig. 10). Alk. Pase at this stage indicated apparent changes; the activity became less intense and decreased in the upper part of the gutters (fig. 2). The upper limits of the reactive epithelia were close to the surviving taste buds. On the 20th postoperative day, taste bud cells had already completely disappeared and were replaced by squamous epithelium. No ATPase activity was observable in the epithelial layer, which became thinner than normal, but subgemmal nerve fibers running along the gutters still gave the definite positive reaction (fig. 7). No discernible difference of ATPase activity was found in the subgemmal nerve plexus of both normal and operated circumvallate RESULTS papillae. Some reaction products of acid Alk. Pase, ATPase and acid Pase were Pase were found scattered in the epithelocalized in specific relationship to the lium covering the bottom of the gutter taste bud as already reported (Iwayama Each group of figures 1, 5 and 9, figures 2, 6 and Nada, '67b) ; alk. Pase was present not in the taste bud but on the superficial layer and 10, figures 3, 7 and 11, and figures 4, 8 and 12 contains photomicrographs of three serial secof the epithelium lining the gutter with tions obtained from the different circumvallate taste buds in the side of circumvallate papillae. All the figures were photographed a t papilla (fig. 1). ATPase and acid Pase, on x 80. Fig. 1 Alk. Pase i n control. The superficial the other hand, were present in the taste layers of the epithelium covering the gutters indibud. The former was associated with the cate a n intense reaction, which is confined to the membrane of taste bud cells (fig. 5) and epithelium with taste buds. The capillary walls in the latter was found in the supranuclear the lamina propria are also responsible for the reaction. cytoplasm (fig. 9). Fig. 2 Alk. Pase seven days after the operaAs shown by previous investigators, in tion. Up to second postoperative day, the activity general, taste buds gradually disappeared is still as strong as in the controls. But the refirst from the upper level in the gutter and action becomes gradually less intense and recedes along the gutters downward. The upper limit of finally were completely lost seven to ten the activity corresponds to the location of the days after the operation. This fact is uppermost surviving taste bud (compare with clearly shown in figures 5, 6 , and 7, which fig. 6). Fig. 3 Alk. Pase 20 days after the operation. illustrate the ATPase reaction. activity becomes very feeble except as a Up to the second day after the operation, The strongly reactive fleck near a n opening of a linthe number of taste bud cells contained in gual gland. The reaction o n the capillary walls a taste bud seemed to decrease but they in the lamina propria is unaltered. The lingual were not counted. In this point, ATPase gland with the activity is shown at the lower left. Fig. 4 Alk. Pase 37 days after the operation. activity was still intense in association with The intense reaction is present along three walls the membranes of taste bud cells, while of two gutters. In the left gutter, the upper limit acid Pase activity was strongly positive in of the activity is higher on the outer wall than all the taste bud cells, but some lacking in o n the inner wall, while the activity on the right wall is negligible. The distribution of the the controls. Alk. Pase was also as strongly inner activity is well correlated with the regenerated reactive as in the normal state. t-ste buds which are shown in figure 8. was ligated to prevent regeneration, while in the animals in which regeneration was anticipated, much care was paid not to impair the proximal portion of the nerve and both stumps were left in situ without any devices. The animals were sacrificed between 1 and 60 days after the operation. The tongue containing the circumvallate papilla was removed from an anesthetized animal with ether. A small piece of specimen was trimmed in order to get suitable sections of the papilla and was directly frozen in isopentane cooled with solid carbon dioxide. The cryostat sections were serially cut in 15 u thickness, dried with an electric fan at room temperature and incubated to demonstrate alk. Pase, ATPase and acid Pase after the method of Burstone ('61), Wachstein and Meisel ('57) and Goldberg and Barka ( ' 6 2 ) respectively, using every third section for the demonstration of each phosphatase activity. HISTOCHEMISTRY OF DE- AND REGENERATING TASTE BUD Figures 1-4 33 34 TAKASHI IWAYAMA AND OSAMI NADA (fig. 11) and might indicate an infiltration of leukocytes (Guth, '57) or reaction artifacts. Alk. Pase became feeble with the exception of intensely reactive speckles near the openings of posterior lingual gland (fig. 3). Further examination up to the 60th day showed no additional loss of alk. Pase activity from the gutters. About 25 days after the operation for regeneration, following the reinnervation of the glossopharyngeal nerve, taste buds began to reappear from the lower portion of the gutter upwards. ATPase activity was strongly positive on the membrane of newly formed taste bud cells in the epithelial layer which had regrown to an original thickness. Following the regeneration of taste buds, alk. Pase again appeared on the gutter wall. Acid Pase, on the other hand, was not distinct yet in the cytoplasm of newly formed taste bud cells. In this experiment, the renewal of the taste buds was rather poor as a result of the poor regeneration of the amputated nerve fibers. Figures 4, 8 and 12 were taken from an animal 37 days after the operation. Several regenerated taste buds were clearly indicated by the staining of ATF'ase in figure 8 where taste buds reappeared higher in the outer wall of the left gutter than in the inner wall, while in the right gutter, the inner wall indicated complete absence of taste buds. These relations were well correlated with the alk. Pase activity in figure 4, that is, the upper limit of the activity was higher on the left outer wall than on the left inner wall and the activity on the right inner wall was negligible. Acid Pase were moderately present in some of taste bud cells of figure 12 and this result indicated that acid Pase of taste bud cells developed somewhat later than the reappearance of taste bud cells. DISCUSSION Alk. Pase of the gustatory epithelia has been histochemically indicated by several authors (Bourne, '48; Baradi and Bourne, '51, '53, '59; Rakhawy, '62; Baradi and Brandes, '63). This enzyme is endowed with important but different roles in taste perception by Baradi and Bourne ('51) and Rakhawy ('62), but the absence of this enzyme on fungiform papilla is unfavorable for their hypothesis (Iwayama and Nada, '67a). Moreover, the superficial layers of the gutter, whcih are going to slough off, have a strong enzymatic reaction. Such strong enzymatic activity is, of course, not produced by inanimate cells like superficial squamous cells. Where can one look out for the source of the alk. Pase of the gutter then? Rakhawy ('62) assumed it might be carried from the lingual toncil by lymphocytes. In the present experiment, alk. Pase activity showed a consistent parallel alteration in accordance with the vicissitudes of taste buds. Therefore, it seems reasonable to assume that almost all alk. Pase found on the desquamating epithelia must originate from taste bud cells. The secretory function of taste buds was already demonstrated by the existence of the dense substance which filled taste pores and the secretory droplets of the same nature in taste bud cells (de Lorenzo, '58, '63; Murray and Murray, '67), although the dense substance bears probably no direct relationship to alk. Pase. The circumvallate and foliate papillae have a considerable number of taste buds and their grooves may prevent the enzyme from being easily removed by saliva. The fungiform papilla, on the other hand, has a single taste bud on its top, and therefore the secretion would be scanty and easily dissolved in saliva. In the taste bud of the fungiform papilla of the rat, however, Farbman ('65) noticed the absence of intracellular secretory droplets which are usually present in taste buds of the circumvallate and foliate Fig. 5 ATPase activity in control. The activity is specifically found on the membrane of taste bud cells. In the lamina propria, a strong activity is present along the nerve fibers running in parallel with the gutter, as well as on the blood vessels. Fig. 6 ATPase seven days after the operation. Taste buds, which are shown with ATPase stain, exist in the lower portions of the gutters only. The reaction is still present on the membrane of all the survived taste bud cells. Fig. 7 ATPase 20 days after the operation. All the taste bud cells have already disappeared and therefore no reaction products are observed in the epithelial layer. But in the lamina propria, the intense activity is still present not only in blood vessels but also in subgemmal nerve plexus. Fig. 8 ATPase 37 days after the operation. Taste buds reappear in the lower parts of the gutters and indicate strong activity of ATPase in the epithelial layer. The poor regeneration of taste bud cells are probably related to poor regeneration of the severed nerve. HISTOCHEMISTRY OF DE- AND REGENERATING TASTE BUD Figures 5-8 35 36 TAKASHI IWAYAMA AND OSAMI NADA Figures 9-12 HISTOCHEMISTRY OF DE- AND REGENERATING TASTE BUD papillae. This difference might coincide with the absence of alk. Pase on the fungiform papilla. A small amount of alk. Pase activity might also be derived from lingual glands, distributed on the trench wall and sometimes condensed around the openings of their excretory ducts (fig. 3 ) . ATPase of taste buds was so specific and intense that this reaction could clearly stain taste bud cells in non-stained squamow epithelia as far as they exist. The activity persisted on the membrane of taste bud cells until they disappeared in degeneration, and reappeared at the beginning of their differentiation in regeneration. During the degeneration and regeneration, the existence of taste bud cells, which was apt to be overlooked in the preparation of hematoxylin-eosin stain, could be easily recognized by this staining. In fact, ATPase-reactive cells can be observed even in the epithelium of new-born rat circumvallate papillae, although Torrey ('40) could not recognize taste buds until the ninth postnatal day. The results will be published elsewhere in detail. In lamina propria, as shown in figure 7, the reaction of ATPase activity was found along the subgemmal nerve plexus of the animal of the twentieth postoperative day and no discernible changes were found on the plexus even 60 days after the operation. This finding presumably demonstrates that most of the activity associated with the nerve fibers Fig. 9 Acid Pase i n control. The activity is more intense i n the taste bud cells than in the other epithelial cells, which also show diffuse reaction, but some of taste bud cells are devoid of the activity. The reaction is localized to the supranuclear cytoplasm of the taste bud cells. Ganglion cells with the reaction are present at the base of the papilla. Fig. 10 Acid Pase seven days after the operation. Taste bud cells apparently diminish i n nurnber, but acid Pase is strongly reactive i n all the surviving taste bud cells, mostly i n their supranuclear regions. Fig. 11 Acid Pase 20 days after the operation. The activity of the epithelial layers shows a weak and diffuse staining. Some reaction products are observed in the epithelium covering the bottom of the gutters and might indicate an infiltration of leukocytes. A n intense staining is present at the lower left, indicating a lingual gland. Fig. 12 Acid Pase 37 days after the operation. Some of the taste bud cells indicate the intense activity of acid Pase in the diffusely reacted epithelium, although the activity is not distinct i n earlier stage. 37 is derived from their Schwann cells, which survive i n their original sites for a long time. Acid Pase was found as one of the lysosoma1 enzymes by de Duve ('55, '63). This enzyme is also reported to be associated with the Golgi apparatus and the secretory granules of the pancreas (Sobel and Avrin, '65; Lazarus et al., '66). As already discussed, taste bud cells seem to have some secretory functions and therefore might have the activity related to secretory function. But taste bud cells, with a life span of 250 & 50 hours (Beidler and Smallman, '65), are usually undergoing degeneration and lysosomes would increase, as a matter of course, in old degenerating cells. In the present experiments, the activity of this enzyme was intense in all the surviving cells after the operation but weak in newly induced ?ells. 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