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Prolonged survival and trophic function of neurons in homologous transplants of sensory ganglion in vivo
EXPERIMENTAL
Prolonged Homologous
NEUROLOGY
Survival
30,
510-524
(1971)
and
Trophic
Transplants
Function
of Sensory
ANDREW
A.
of
Neurons
Ganglion
in Vivo
ZALEWSKI
Laboratory of Newopathology and Neuroamto~l~ical Scimres, National Imtitutr of Newological Diseases a,zd Stroke. Natiolzal Imtitzttes of Health. Pzhlic Health Scvvicc. U.S. Department of Health, Education, and CVelfare, Bctkesda, Maryland Received
in
Novencbrv
20014
10, 1970
Neurons in gustatory ganglia which survive autologous transplanatation ire zia~o retain their trophic effectiveness and can cause taste bud regeneration. In the present study homologous ganglion transplanatation Gt zjivo was performed to see if these neurons would also survive and induce bud regeneration. The vagus nodose ganglion was transplanted in adult male rats and, 30 weeks later, studied for the presence of neurons, the nerves for nerve fiber growth, and the tongue’s vallate papilla for taste buds. A small number of neurons was found in all nine transplanted ganglia ; these neurons appeared morphologically and histochemically normal. Furthermore, some of these surviving neurons gave rise to nerve fibers which caused taste bud regeneration; after removal of the ganglion these buds degenerated. A mild cellular reaction consisting of large-nucleated cells, neutrophilic leucocytes, and small lymphocytes was present around but not within the transplanted ganglia and its nerve fibers; a cellular reaction was not seen in the papilla. The results demonstrate that neurons can survive homologous transplantation ill zrizlo and still perform a trophic function, and it therefore seems that neurons may not evoke, or be adversly affected, by an immune reaction. Introduction
The development, maintenance. and regeneration of taste buds requires the continuous trophic influence of the intact gustatory neuron (7, 18, 22). Since a previous stud\- demonstrated that neurons in autologous transplants of gustatory ganglia in z&fo retained their trophic effectiveness (Zl), the present experiment was performed to see if in viva homologously transplanted gustatory neurons would also cause bud regeneration. Taste buds in the vallate papilla of the rat’s tongue are innervated by the glossopharyngeal nerves and following transection of these nerves the buds disappear (6. 18). Since buds reappear after reinnervation by nerve fibers from the vagal nodose ganglion (19), this ganglion was selected for transplantation. By performing homologous transplantation in z~iz~o it was hoped to determiue whether the immunological system would permit 510
GANGLION
homologous neuron survival. nerve fiber growth, tion (i.e., taste bud regeneration) .
Materials
511
TRANSPLANTS
and
and trophic
nerve func-
Methods
Osborne-Mendel male rats were anesthetized with chloral hydrate (40 mg/lOO g, ip), and in the host animal (200-250 g), the glossopharyngeal nerve transected bilaterally beneath the stylohyoid muscle and the central stump of the nerves buried into adjacent neck muscle in order to prevent its regeneration back to the tongue. The left nodose ganglion was taken from a donor animal (250-300 g), and the peripheral fibers of the three main nerve trunks of the ganglion joined unilaterally to the left distal glossopharyngeal nerve trunk of the host. Nerve anastomosis was performed by tucking and aligning the ends of the nerves into a sleeve of arterial graft (18). These animals (along with control rats having a normally innervated or denervated papilla ) were allowed to survive 30 weeks at which time four animals from each group were studied. In addition, the acute effects of denervation on the taste buds of normal control rats were compared with five transplant reinnervated papillae that were denervated after 30 weeks and examined 5 days later. At the appropriate time, normal and transplanted nodose ganglia were between muscle to facilitate sectioning, and frozen removed, “sandwiched” in liquid nitrogen. Thick frozen sections (8 p) were cut and the various sections stained with PAS-hematoxylin or thionine, or incubated to detect acid (4) or alkaline (3) phosphatase, adenosine triphosphatase (17)) succinic dehydrogenase (12), or cholinesterase (5) enzyme activity. In one transplanted ganglion, every fourth section of the ganglion was mounted and stained with PAS-hematoxylin. A quantitative count of the neurons was compared with that from a similarly prepared normal left nodose ganglion. The site of the nerve anastomosis, together with the left vagus and glossopharyngeal nerves from unoperated rats were sectioned and stained with PAS-hematoxylin or for cholinesterase activity. After incubation for cholinesterase, nerve sections were treated with silver nitrate in order to intensify the sulfide deposits at the cholinesterase active sites (8). After removal of the ganglia and nerves. the animal was exsanguinated and the tongue portion containing the vallate papilla frozen in Dry Ice-cooled isopentane. Frozen serial cross sections (6 p) were cut and incubated to detect adenosine triphosphatase or cholinesterase activity. Control sections for all enzymes were incubated in a solution lacking added substrate. After incubation, acid and alkaline phosphatase, and adenosine triphosphatase slides were counterstained with methyl green while all cholinesterase slides were stained with hematoxylin.
512
ZALEWSKI
Results
Gross Obscwations of the Animal, Tramplanted Ganglia, and Nerve Auastomosis Site. Kane of the operations performed had any ill effect on the growth or appearance of the animal. In all nine animals, the transplanted ganglion was found, and it appeared dull gray in color. It was not particularly adherent to surrounding tissue or enclosed by large amounts of fibrous tissue. Small, fine nerve strands had grown out of the central end of the ganglion, but these were not traced any great distance nor examined histologically. The two nerves had united within the arterial graft, and nerve trunks on either side of the graft appeared slightly white. In five instances some nerve fibers grew out around rather than into the original distal glossopharyngeal nerve trunk and apparently ramified locally. Since at the operation all three nerve trunks of the nodose ganglion were placed into the graft and since only one glossopharyngeal trunk was available into which the vagal fibers could grow, this growth of regenerating fibers outside the glossopharyngeal nerve trunk was expected. The central stumps of the glossopharyngeal nerves were identified beneath the stylohoid muscle still embedded in neck muscle and at no time were nerve fibers seen growing out to contaminate the transplanted ganglion or nerve anastomosis site. X~urons in thr 90~1ml amf Tmmplanted Nodosc Gaqlion. The presence and distribution of neurons in the ganglion was revealed by all the histochemical stains. The PAS-hematoxylin stain, however, was used for most descriptive and quantitative purposes. In the normal ganglion, the neurons were located mainly at the periphery of the ganglion with some also being interspersed in the core (Figs. 1A and 2A). The remainder of the ganglion was filled by nerve fibers which at various levels occupied the central or peripheral parts (Fig. IA). Small, medium, and larger sized neurons were present throughout, and most had a centrally located nucleus and similar distribution of Niss! su!,stance and enzymes. ,4lthough the distribution of enzymes was similar in all cells, activity differed greatly with simi!ar sized cells having mild to intense enzyme activity. Thioninestained sections showed coarse clumps of Nissl substance variously distributed in the cytoplasm of the cell body with the region around the nucleus showing the most intense reaction (Fig. 2A). Acid phosphatase
FIG. 1. A. Normal nodose ganglion. Neurons are found interspersed among the PAS myelin stained nerve fibers. B. Thirty-week homologously transplanted nodose ganglion. The number of neurons present (arrows outline groups of neurons) is reduced, and they are located mainly at the periphery of the ganglion. PAS-stained myelinated nerve fibers are present, and note the cellular reaction around but not in the ganglion. PAS-hematoxylin stain. X 130.
514
ZALEWSKI
.
A .
‘c >
.
‘.
_
-..
FIG. 2. A-C. Thionine stain. -4. Normal throughout the ganglion and have a perinuclear C. Thirty-week homologously transplanted ( X700). Surviving neurons are present at neurons like the one indicated by the arrow substance (C). D and E. Acid phosphatase (D) and transplanted (E) ganglion have a tion of enzyme activity.
nodose ganaglion. Neurons are present distribution of Nissl substance. B and ganglion (B) ( X 70) and neuron (C) the periphery of the ganglion, and many in B have a normal distribution of Nissl activity ( X300). Neurons in the normal similar intensity and perinuclear distribu-
GANGLION
TRANSPLANTS
515
activity was present in the form of coarse irregular granules in primarily a perinuclear distribution (Fig. 2D). Alkaline phosphatase and adenosine triphosphatase activity was present at the cell border with only occasional sites of activity being present within the neuron. Fine granules which were homogenously distributed throughout the cytoplasm indicated the sites of succinic dehydrogenase activity. Cholinesterase activity was diffused in the cytoplasm of the neurons and was especially intense in some of the small sized ones. All sections of ganglia, nerves, and papillae that were incubated without added substrate failed to reveal enzyme staining. All nine transplanted ganglia contained neurons, and it was clearly evident that the number of neurons present was markedly reduced. Almost all surviving neurons were peripherally located (Figs. 1B and 2B) with only an occasional few being present in the center of the ganglion. Neuron counts in one transplanted ganglia revealed 606 neurons present in contrast to the 2578 cells observed in a normal ganglion. Nerve fibers were within the transplanted ganglia, but they occupied less space than in a normal ganglion. All three sizes of neurons were present in the transplanted ganglia, but no quantitative count of each type was made. Some neurons had a normal amount and distribution of Nissl substance (Fig. 2C) while in others it was sparsely distributed. Acid phosphatase activity appeared normal both in distribution and intensity (Fig. 2E). Alkaline phosphatase activity at the cell borders generally was slightly reduced, but adenosine triphosphatase appeared normal as did the cholinesterase activity within the cytoplasm. Nerve Fibers in the Transplant
the Normal Vagus and Glossopharyngeal Nerzle and Nerve Anastomosis. Nerve fibers in the peripheral nerves
in
were studied with the PAS-hematosylin and cholinesterase-silver stains. In frozen sections PAS stains a myelin component whereas cholinesterase stains the nerve membrane and cytoplasm. Silver treatment of the cholinesterase sections subsequently causes the nerve fiber to appear as a black dot in cross sections or as a wavy line in longitudinal sections. Nerve fiber stains were used to detect the presence of nerve fibers and not to make quantitative counts. In cross sections of both normal nerves, PAS staining of nerve fibers appears as smooth rings surrounding central clear areas while in cholinesterase sections fibers appear as black dots, and depending on whether the fiber is adequately myelinated surrounded by a clear halo. In longitudinal sections, the nerve fibers appeared as straight or undulating lines. Many PAS- and cholinesterase-silver-stained nerve fibers were present in all the transplanted ganglia (Fig-. 1B) and in all three nodose nerve trunks (Fig. 3A). Sections of nerve at the distal glossopharyngeal end of the arterial graft (Fig. 3B) and beyond revealed PAS- and cholinesterase-stained nerve fibers demonstrating clearly that the trans-
516
ZALEWSKI
FIG. 3. Cholinesterase-silver stain. X130. A. Peripheral nerve trunks of the vagal nodose ganglion 30 weeks after homologous transplantation. Numerous nerve fibers are present in the nerve trunks, and these fibers appear as black dots in cross section and as undulating lines in oblique or longitudinal sections. Note the cellular reaction
GANGLION
planted nodose neurons successfully byond the nerve anastomosis site.
517
TRANSPLANTS
regenerated
their
fibers across
and
Taste Buds in the Normal and Glossopharyngeal Denervated Papilla and in the Transplanted Nodose Ganglion Reinnervated and Denervated Papilla. Taste buds were identified by their adenosine triphosphatase ac-
tivity and their associated innervation was revealed by nerve fiber cholinesterase staining. In the normal glossopharyngeal innervated papilla, taste buds are found in the epithelium of the trench walls (Fig. 4A). Each normal bud extends through the entire width of the epithelium, and the taste cells exhibit intense membrane adenosine triphosphatase activity (Fig. 5A). An intense nerve fiber cholinesterase reaction is present in the connective tissue immediately beneath the epithelium where the taste buds are located (Fig. 5H) . Taste buds do not disappear at the same time or in the same pattern after nerve transection and so a variety of forms can be present depending on the denervation interval studied (6, IS). The S-day denervated taste bud interval was chosen for the present study becauseby this time previous studies have demonstrated (l&20) that no normal buds or patterns of nerve innervation are present. Most taste buds had disappeared 5 days after bilateral glossopharyngeal nerve transection (Fig. 4B), and those which remained were smaller than normal, contained fewer taste cells, and had left the basement membrane and were moving toward the epithelial surface (Figs. 4B and 5B). The degenerating taste bud cells still possessed intense membrane adenosine triphosphatase activity and so their location and course of movement in the epithelium were readily recognized. All traces of taste buds were gone by 10 days (Fig. 4C). No buds were found in the papillae that had been chronically denervated for 30 weeks, and in these specimensthe epithelium was markedly atrophied (Fig. SC). Nerve fiber cholinesterase activity beneath the taste buds was lost 5 days after denervation and remained undetectable at 10 days or 30 weeks postoperatively. Taste buds were present in three of the four papillae examined 30 weeks after nodose ganglion transplantation (Fig. 4D). In one papilla 12 buds were found while the others had 14 and 19; these are far fewer than the hundreds of buds normally present in the papilla (6). The regenerated around the transplanted nerve trunks which consists of large-nucleated cells (? macrophages) (arrow, m), neutrophilic leucocytes (arrow, n), and small lymphocytes (arrow, L). B. Glossopharyngeal nerve end of the arterial graft anastomosis site. Many nerve fibers from the nodose ganglion have grown into the distal glossopharyngeal nerve trunk (arrow, gt) while other vagal fibers (unlabeled arrows) have regenerated around it.
ZALEWSKI
X70. -4. Normal papilla. Numerous taste FIG. 4. Adenosine triphosphatase activity. buds are present in the epithelium of the trench walls. B. Five-day glossopharyngeal nerve denervated papilla. The number of taste buds is reduced, and those buds present are in various stages of degeneration. C. Ten-day glossopharyngeal nerve denervated papilla. All taste buds have disappeared. D. Thirty-week homologous nodose ganglion transplant reinnervated papilla. Regenerated taste buds are randomly located in the trench walls.
GANGLION
TRANSPLANTS
519
FIG. 5. A-G. Adenosine triphosphatase activity. A. Normal trench wall. Normal buds extend through the entire width of the epithelium, and all the taste cells exhibit intense enzyme activity. X 300. El. Five-day glossopharyngeal nerve denervated trench wall. Degenerating buds have moved away from the basement membrane toward the epithelial surface and they are smaller than normal and contain fewer taste cells. X300. C. Thirty-week glossopharyngeal nerve denervated trench wall. No taste buds are present, and the trench wall epithelium is atrophied. X300. D. Thirty-week homoogous nodose transplant reinnervated trench wall. The regenerated bud, like normal ones, extends through the entire width of the epithelium and has numerous taste ce!ls with intense adenosine triphophatase activity. X 300. E-G. Thirty-week homologous nodose ganglion transplant reinnervated and subsequently 5-day denervated papilla (E) ( X 70) and trench walls (F and G) ( X 300). Two degenerating buds are seen in the epithelium of the trench walls. H-K. Cholinesterase activity. X300. An intense nerve fiber cholinesterase reaction is present immediately beneath the epithlium where a normal glossopharyngeal innervated bud is located (arrow) (H). Enzyme activity is also present beneath the ganglion transplant reinnervated bud (arrow) (I), but it disappears after removing the ganglion within 5 days from beneath the degenerating buds (arrow) (J and K).
520
ZALEWSKI
buds were randomly located in the trench wall epithelium, and each estended the full width of the epithelium with taste cells showing intense adenosine triphosphatase activity (Fig. SD). A moderate cholinesterase reaction was present beneath the epithelium where the regenerated buds were located (Fig. 51). Only occasional tracs of cholinesterase activity were seen near the epithelium of the one papilla in which no taste buds were found. Degenerating buds were found in three of the five papilla studied 5 days after denervating the ganglion transplant reinnervated papilla (Fig. 5E). These buds, as in the previously described 5-day glossopharyngeal denervated papilla, were small, had few cells, and were present only near the surface of the trench wall (Fig. 5F and G). No cholinesterase activity was seen beneath these clegenerating buds (Fig. 5J and K). The remaining two papillae lacked taste buds, and it is therefore not certain whether buds were present prior to the denervation or whether all buds had disappeared within the 5-day postoperative interval.
Cellular Response to the Hovlzologously Transplanted Nodose Ganglia and Nerve Fibers. A mild cellular reaction of large-nucleated cells, neutrophilic leucocytes, and small lymphocytes was present around but not in the transplanted nodose ganglia (Fig. IB) and its nerve trunks (Fig. 3A). The latter two cell types were identified because they gave the same type of cholinesterase stain when they were compared to peripheral blood. No such cellular reaction was seen in the distal glossopharyngeal nerve trunk into which the vagal nerve fibers had regenerated or in the papilla.
Observations Concerning Vascularization of the Transplanted Nodose Ga,ngZion. No direct vascular tracer studies of the transplanted ganglia were performed, but in the ganglia some neutrophilic leucocytes were found. These cells were clearly identified because they are the only normal leucocytes that stain for alkaline pbosphatase activity (16, 18). These leucocytes were few in number and generally found near capillary endothelium (which also stains intensely for alkaline phosphatase). It seems unlikely that these cells represent donor rather than host leucocytes because neutrophilic leucocytes have a lo-14-day life-span, and any donor leucocytes which may have been present in the ganglion at the time of transplantation should have disappeared after 30 weeks. Discussion
The histological studies show that some neurons in the homologously transplanted vagal nodose ganglion survived and that they exhibited a morphology and enzyme activity and distribution similar to that of normal nodose neurons. Other studies of transplanted sensory ganglia have also indicated that some neurons can survive homologous transplantation and,
GANGLION
TRANSPLANTS
521
after undergoing an initial chromatolytic reaction, return to a normal morphological appearance ( 13, 15). However, since transplantation in these studies was made into the brain (13, 15) there was little opportunity to determine if nerve fiber growth or functional reinnervation occurred. In the present study, the neurons not only survived, but some of them regenerated their nerve fibers to the tongue and caused taste bud regeneration. The only other system where transplantd ganglion neurons have been demonstrated to perform a trophic function concerns studies on limb regeneration in the amphibian. In this study it was shown that homologous transplants of spinal ganglia could sustain blastemal growth during the critical period when growth was dependent on the nerve (9). Furthermore, a recent study has demonstrated the chemical nature of this neurotrophic influence by showing that infusions of nerve homogenates will stimulate protein synthesis in the regenerating blastema (10). However, it is uncertain whether the neurons in the transplanted ganglion maintained blastema growth (9) in a physiological manner or whether dead or dying neurons merely liberated a biologically active chemical substance into the blastema for either process seemingly could function as an infused nerve homogenate. This question might be resolved by an experiment in which the ganglia are transplanted several months prior to amputation of the limb; if the limb regenerated it would show that the neurons can exert their tropbic function even after prolonged periods of transplantation. Since taste bud cells are known to undergo a physiological turnover (2)) it appears certain from the results of the present study that the transplanted neurons are performing a trophic function by a physiological mechanism. The fact that the transplanted neurons retained their trophic function does not mean that all aspects of neuronal or taste bud metabolism are normal. For example, impulse conduction, other aspects of neuron morphology or histochemistry. or taste responses might be abnormal, especially since it is highly probable that not all neurons grew out fibers that reinnervated appropriate end organs. Consequently these parameters must be further investigated before the transplanted neurons or regenerated taste buds can be considered fully normal. Neuronal metabolism, in fact, can be altered without the loss of trophic function for it has been shown that chromatolyzed gustatory neurons still can support taste buds (20). The presence of degenerating buds 5 days after transection of the regenerated nodose nerve fibers further demonstrates that the transplanted neurons had indeed performed their trophic function, for in all regards, the degenerating buds resembled those seen after acute transection of the normally glossopharyngeal innervated papilla. Inasmuch as the previous studies of homogously transplanted ganglia
522
ZALE\TSRI
(9, 13, 15) were performed before the basic principles of immunological tissue tolerance were known, it is understandable why no mention was made of cellular reaction to the transplants. The cellular response to the ganglion transplant was, however, an important immunoiogical question that was asked and described in the present study. It should be understood that in order to elicit an immune response the transplanted tissue must be sufficiently antigenic (qualitatively and quantitatively‘) to elicit an immune response, the antigen must enter the lymphatic tissue and stimulate antibody production to it, the antibodies must have a vascular means of getting to the source of the antigen, and the antibody must have the opportunity to freely interact with the antigenic tissue (11). Brain homografts (e.g.. cortex, cerebellum, corpus callosum) have heen reported to induce sensitization inasmuch as skin grafts performed several weeks later are rejected at an accelerated rate (1). However, in this study, no neurons survived transplantation so that antigenicity of the brain tissue was attributed to surviving glial tissue. Before neurons can be studied for antigenicity, transplantation conditions must be designed to promote vascularization so that anoxia can be eliminated as a cause of neuron death. This has been accomplished in homologous heart transplantation studies in which vascular anastomosis is readily accomplished. Early time studies of neurons in intrinsic heart autonomic ganglia of dogs showed histologically normal neurons that were surrounded by a cellular reaction similar to that which occurred around the heart muscle fibers at the time of cardiac failure (14). It was suggested, as in the previous study (1). that the nonneuronal elements of nerve tissue (i.e., neuroglia, myelin. neurilemma cells) were the strongly antigenic component. In the present study neurons survived, regenerated their nerve fibers, and performed a trophic function despite the presence of a cellular response. The total numbr of neurons surviving chronic homologous transplantation did not appear different from the number seen after antologous transplantation in ZGZV (21). It seems therefore that neuron death in the homologously transplanted ganglia was due to some transplantation shock rather than an immune response. A notable difference between the cardiac and ganglion transplants is that in the former, a cellular rejection reaction occurred around the neurons in the ganglia. In the nodose ganglion it was present around the ganglion capsule and connective tissue of the vagal nerve trunks but not in the ganglion core where the neurons are located nor in the fascicles containing the nerve fibers. Further investigations are needed to evaluate the antigenicity of neurons. These studies must employ highly imbred but antigenically incompatible animals as donors and hosts rather than the random-bred unmatched animals used in the present study. First- and second-set rejection studies must
GANGLION
TRANSPLANTS
523
also be included to determine whether neurons possessno immunologically competent antigen, a unique antigen, or a common tissue antigen. It is clear though that some neurons in homologous ganglion transplants can survive and trophically function for prolonged periods. References 1. BARNES, A. D. 1969. The immunogenicity of brain, ovary, and skin allografts in mice. Transfilantation 8 : 379-382. 2. BEIDLER, L. M.. and R. L. SMALLXXAN. 1965. Renewal of cells within taste buds. J. Cell Biol. 27 : 263-272. 3. BURSTONE, M. S. 1961. Histochemical demonstration of phosphatases in frozen sections with Naphtha1 AS-phosphates. J. Histochem. Cytockem. 9 : 146156. 4. GOLDBERG, A. F., and T. BARKA. 1962. Acid phosphatase activity in human blood cells. Nature London 195 : 297. 5. G~MORI, G. 1952. “Microscopic Histochemistry : Principles and Practice.” Univer. of Chicago Press, Chicago, Ill. 6. GUTH, L. 1957. The effects of glossopharyngeal nerve transection on the circumvallate papilla of the rat. Anat. Rec. 129 : 715-731. 7. GUTH, L. 1969. Trophic effects of vertebrate neurons. Neurosci. Res. Program Bull. 7 : l-73. 8. HENDERSON, J. R. 1967. The use of silver for intensifying sulfide deposits in the cholinesterase technique. Stain Tecknol. 42 : 101-102. 9. KAMRIN, A. A., and M. SINGER. 1959. The growth influence of spinal ganglia implanted into the denervated forelimb regenerate of the newt, Triturus. J. Morjkol. 225 : 82M27. 10. LIEBOWITZ. P., and M. SINGER. 1970. Neurotrophic control of protein synthesis in the regenerating limb of the newt, Triturus. Nature London 225: 824-827. 11. MEDAWAR, P. B. 1948. Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Brit. J. Exp. Patkol. 29 : 5869. 12. PEARSE, A. G. E. 1961. Methods (#2) for succinate dehydrogenase using MTT, p. 910. In “Histochemistry, Theoretical, and Applied,” 2nd ed. Little, Brown, Boston. 13. RANSON, S. W. 1914. Transplantation of the spinal ganglion with observations on the significance of the complex types of spinal ganglion cells. J. Cofnp. Neural. 24 : 547-558. 14. SHARDEY, G. C., E. COOPER, and W. G. R. M. DE BOER. 1970. Differential rejection of neurones and neuroglia in canine cardiac allografts. Nature London 228: 69-71. 15. TIDD, C. W. 1932. The transplantation of spinal ganglia in the white rat. A study of the morphological changes in surviving cells. J. Co”zp. Neural. 55: 531-543. 16. TITTOBELLO, A., and A. AGOSTONI. 1967. Leucoycyte alkaline phosphatase: A quantitative and qualitative study in normal and leukemic cells. Enzyvmol. Biol. C&z. 8 : 413420. 17. WACHSTEIN, M., and E. MEISEI.. 1957. Histochemistry of hepatic phosphatase at a physiologic pH. Amer. J. Clin. Pathol. 27 : 13-23. 18. ZALEWSKI, A. A. 1969a. Combined effects of testosterone and motor, sensory, or gustatory reinnervation on the regeneration of taste buds in the rat. E.rp. Neural. 24 : 285-297.
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ZALEWSKI
A. A. 1969b. Regeneration of taste buds after reinnervation by perior central fibers of vagal ganglia. Exp. Neural. 25 : 429-437. 20. ZALEWSKI, A. A. 1970. Continuous trophic influence of chromatolyzed gustatory neurons on taste buds. Aeat. Rec. 167 : 165-173. 21. ZALEWSKI, A. ‘4. 1970. Trophic influence of irk Cm transplanted sensory neurons on taste buds. Exb. h’clruol. 29: 462-467. 19.
Z.~LE~VSKI,
pheral
Prolonged Homologous
NEUROLOGY
Survival
30,
510-524
(1971)
and
Trophic
Transplants
Function
of Sensory
ANDREW
A.
of
Neurons
Ganglion
in Vivo
ZALEWSKI
Laboratory of Newopathology and Neuroamto~l~ical Scimres, National Imtitutr of Newological Diseases a,zd Stroke. Natiolzal Imtitzttes of Health. Pzhlic Health Scvvicc. U.S. Department of Health, Education, and CVelfare, Bctkesda, Maryland Received
in
Novencbrv
20014
10, 1970
Neurons in gustatory ganglia which survive autologous transplanatation ire zia~o retain their trophic effectiveness and can cause taste bud regeneration. In the present study homologous ganglion transplanatation Gt zjivo was performed to see if these neurons would also survive and induce bud regeneration. The vagus nodose ganglion was transplanted in adult male rats and, 30 weeks later, studied for the presence of neurons, the nerves for nerve fiber growth, and the tongue’s vallate papilla for taste buds. A small number of neurons was found in all nine transplanted ganglia ; these neurons appeared morphologically and histochemically normal. Furthermore, some of these surviving neurons gave rise to nerve fibers which caused taste bud regeneration; after removal of the ganglion these buds degenerated. A mild cellular reaction consisting of large-nucleated cells, neutrophilic leucocytes, and small lymphocytes was present around but not within the transplanted ganglia and its nerve fibers; a cellular reaction was not seen in the papilla. The results demonstrate that neurons can survive homologous transplantation ill zrizlo and still perform a trophic function, and it therefore seems that neurons may not evoke, or be adversly affected, by an immune reaction. Introduction
The development, maintenance. and regeneration of taste buds requires the continuous trophic influence of the intact gustatory neuron (7, 18, 22). Since a previous stud\- demonstrated that neurons in autologous transplants of gustatory ganglia in z&fo retained their trophic effectiveness (Zl), the present experiment was performed to see if in viva homologously transplanted gustatory neurons would also cause bud regeneration. Taste buds in the vallate papilla of the rat’s tongue are innervated by the glossopharyngeal nerves and following transection of these nerves the buds disappear (6. 18). Since buds reappear after reinnervation by nerve fibers from the vagal nodose ganglion (19), this ganglion was selected for transplantation. By performing homologous transplantation in z~iz~o it was hoped to determiue whether the immunological system would permit 510
GANGLION
homologous neuron survival. nerve fiber growth, tion (i.e., taste bud regeneration) .
Materials
511
TRANSPLANTS
and
and trophic
nerve func-
Methods
Osborne-Mendel male rats were anesthetized with chloral hydrate (40 mg/lOO g, ip), and in the host animal (200-250 g), the glossopharyngeal nerve transected bilaterally beneath the stylohyoid muscle and the central stump of the nerves buried into adjacent neck muscle in order to prevent its regeneration back to the tongue. The left nodose ganglion was taken from a donor animal (250-300 g), and the peripheral fibers of the three main nerve trunks of the ganglion joined unilaterally to the left distal glossopharyngeal nerve trunk of the host. Nerve anastomosis was performed by tucking and aligning the ends of the nerves into a sleeve of arterial graft (18). These animals (along with control rats having a normally innervated or denervated papilla ) were allowed to survive 30 weeks at which time four animals from each group were studied. In addition, the acute effects of denervation on the taste buds of normal control rats were compared with five transplant reinnervated papillae that were denervated after 30 weeks and examined 5 days later. At the appropriate time, normal and transplanted nodose ganglia were between muscle to facilitate sectioning, and frozen removed, “sandwiched” in liquid nitrogen. Thick frozen sections (8 p) were cut and the various sections stained with PAS-hematoxylin or thionine, or incubated to detect acid (4) or alkaline (3) phosphatase, adenosine triphosphatase (17)) succinic dehydrogenase (12), or cholinesterase (5) enzyme activity. In one transplanted ganglion, every fourth section of the ganglion was mounted and stained with PAS-hematoxylin. A quantitative count of the neurons was compared with that from a similarly prepared normal left nodose ganglion. The site of the nerve anastomosis, together with the left vagus and glossopharyngeal nerves from unoperated rats were sectioned and stained with PAS-hematoxylin or for cholinesterase activity. After incubation for cholinesterase, nerve sections were treated with silver nitrate in order to intensify the sulfide deposits at the cholinesterase active sites (8). After removal of the ganglia and nerves. the animal was exsanguinated and the tongue portion containing the vallate papilla frozen in Dry Ice-cooled isopentane. Frozen serial cross sections (6 p) were cut and incubated to detect adenosine triphosphatase or cholinesterase activity. Control sections for all enzymes were incubated in a solution lacking added substrate. After incubation, acid and alkaline phosphatase, and adenosine triphosphatase slides were counterstained with methyl green while all cholinesterase slides were stained with hematoxylin.
512
ZALEWSKI
Results
Gross Obscwations of the Animal, Tramplanted Ganglia, and Nerve Auastomosis Site. Kane of the operations performed had any ill effect on the growth or appearance of the animal. In all nine animals, the transplanted ganglion was found, and it appeared dull gray in color. It was not particularly adherent to surrounding tissue or enclosed by large amounts of fibrous tissue. Small, fine nerve strands had grown out of the central end of the ganglion, but these were not traced any great distance nor examined histologically. The two nerves had united within the arterial graft, and nerve trunks on either side of the graft appeared slightly white. In five instances some nerve fibers grew out around rather than into the original distal glossopharyngeal nerve trunk and apparently ramified locally. Since at the operation all three nerve trunks of the nodose ganglion were placed into the graft and since only one glossopharyngeal trunk was available into which the vagal fibers could grow, this growth of regenerating fibers outside the glossopharyngeal nerve trunk was expected. The central stumps of the glossopharyngeal nerves were identified beneath the stylohoid muscle still embedded in neck muscle and at no time were nerve fibers seen growing out to contaminate the transplanted ganglion or nerve anastomosis site. X~urons in thr 90~1ml amf Tmmplanted Nodosc Gaqlion. The presence and distribution of neurons in the ganglion was revealed by all the histochemical stains. The PAS-hematoxylin stain, however, was used for most descriptive and quantitative purposes. In the normal ganglion, the neurons were located mainly at the periphery of the ganglion with some also being interspersed in the core (Figs. 1A and 2A). The remainder of the ganglion was filled by nerve fibers which at various levels occupied the central or peripheral parts (Fig. IA). Small, medium, and larger sized neurons were present throughout, and most had a centrally located nucleus and similar distribution of Niss! su!,stance and enzymes. ,4lthough the distribution of enzymes was similar in all cells, activity differed greatly with simi!ar sized cells having mild to intense enzyme activity. Thioninestained sections showed coarse clumps of Nissl substance variously distributed in the cytoplasm of the cell body with the region around the nucleus showing the most intense reaction (Fig. 2A). Acid phosphatase
FIG. 1. A. Normal nodose ganglion. Neurons are found interspersed among the PAS myelin stained nerve fibers. B. Thirty-week homologously transplanted nodose ganglion. The number of neurons present (arrows outline groups of neurons) is reduced, and they are located mainly at the periphery of the ganglion. PAS-stained myelinated nerve fibers are present, and note the cellular reaction around but not in the ganglion. PAS-hematoxylin stain. X 130.
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A .
‘c >
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_
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FIG. 2. A-C. Thionine stain. -4. Normal throughout the ganglion and have a perinuclear C. Thirty-week homologously transplanted ( X700). Surviving neurons are present at neurons like the one indicated by the arrow substance (C). D and E. Acid phosphatase (D) and transplanted (E) ganglion have a tion of enzyme activity.
nodose ganaglion. Neurons are present distribution of Nissl substance. B and ganglion (B) ( X 70) and neuron (C) the periphery of the ganglion, and many in B have a normal distribution of Nissl activity ( X300). Neurons in the normal similar intensity and perinuclear distribu-
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activity was present in the form of coarse irregular granules in primarily a perinuclear distribution (Fig. 2D). Alkaline phosphatase and adenosine triphosphatase activity was present at the cell border with only occasional sites of activity being present within the neuron. Fine granules which were homogenously distributed throughout the cytoplasm indicated the sites of succinic dehydrogenase activity. Cholinesterase activity was diffused in the cytoplasm of the neurons and was especially intense in some of the small sized ones. All sections of ganglia, nerves, and papillae that were incubated without added substrate failed to reveal enzyme staining. All nine transplanted ganglia contained neurons, and it was clearly evident that the number of neurons present was markedly reduced. Almost all surviving neurons were peripherally located (Figs. 1B and 2B) with only an occasional few being present in the center of the ganglion. Neuron counts in one transplanted ganglia revealed 606 neurons present in contrast to the 2578 cells observed in a normal ganglion. Nerve fibers were within the transplanted ganglia, but they occupied less space than in a normal ganglion. All three sizes of neurons were present in the transplanted ganglia, but no quantitative count of each type was made. Some neurons had a normal amount and distribution of Nissl substance (Fig. 2C) while in others it was sparsely distributed. Acid phosphatase activity appeared normal both in distribution and intensity (Fig. 2E). Alkaline phosphatase activity at the cell borders generally was slightly reduced, but adenosine triphosphatase appeared normal as did the cholinesterase activity within the cytoplasm. Nerve Fibers in the Transplant
the Normal Vagus and Glossopharyngeal Nerzle and Nerve Anastomosis. Nerve fibers in the peripheral nerves
in
were studied with the PAS-hematosylin and cholinesterase-silver stains. In frozen sections PAS stains a myelin component whereas cholinesterase stains the nerve membrane and cytoplasm. Silver treatment of the cholinesterase sections subsequently causes the nerve fiber to appear as a black dot in cross sections or as a wavy line in longitudinal sections. Nerve fiber stains were used to detect the presence of nerve fibers and not to make quantitative counts. In cross sections of both normal nerves, PAS staining of nerve fibers appears as smooth rings surrounding central clear areas while in cholinesterase sections fibers appear as black dots, and depending on whether the fiber is adequately myelinated surrounded by a clear halo. In longitudinal sections, the nerve fibers appeared as straight or undulating lines. Many PAS- and cholinesterase-silver-stained nerve fibers were present in all the transplanted ganglia (Fig-. 1B) and in all three nodose nerve trunks (Fig. 3A). Sections of nerve at the distal glossopharyngeal end of the arterial graft (Fig. 3B) and beyond revealed PAS- and cholinesterase-stained nerve fibers demonstrating clearly that the trans-
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FIG. 3. Cholinesterase-silver stain. X130. A. Peripheral nerve trunks of the vagal nodose ganglion 30 weeks after homologous transplantation. Numerous nerve fibers are present in the nerve trunks, and these fibers appear as black dots in cross section and as undulating lines in oblique or longitudinal sections. Note the cellular reaction
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planted nodose neurons successfully byond the nerve anastomosis site.
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their
fibers across
and
Taste Buds in the Normal and Glossopharyngeal Denervated Papilla and in the Transplanted Nodose Ganglion Reinnervated and Denervated Papilla. Taste buds were identified by their adenosine triphosphatase ac-
tivity and their associated innervation was revealed by nerve fiber cholinesterase staining. In the normal glossopharyngeal innervated papilla, taste buds are found in the epithelium of the trench walls (Fig. 4A). Each normal bud extends through the entire width of the epithelium, and the taste cells exhibit intense membrane adenosine triphosphatase activity (Fig. 5A). An intense nerve fiber cholinesterase reaction is present in the connective tissue immediately beneath the epithelium where the taste buds are located (Fig. 5H) . Taste buds do not disappear at the same time or in the same pattern after nerve transection and so a variety of forms can be present depending on the denervation interval studied (6, IS). The S-day denervated taste bud interval was chosen for the present study becauseby this time previous studies have demonstrated (l&20) that no normal buds or patterns of nerve innervation are present. Most taste buds had disappeared 5 days after bilateral glossopharyngeal nerve transection (Fig. 4B), and those which remained were smaller than normal, contained fewer taste cells, and had left the basement membrane and were moving toward the epithelial surface (Figs. 4B and 5B). The degenerating taste bud cells still possessed intense membrane adenosine triphosphatase activity and so their location and course of movement in the epithelium were readily recognized. All traces of taste buds were gone by 10 days (Fig. 4C). No buds were found in the papillae that had been chronically denervated for 30 weeks, and in these specimensthe epithelium was markedly atrophied (Fig. SC). Nerve fiber cholinesterase activity beneath the taste buds was lost 5 days after denervation and remained undetectable at 10 days or 30 weeks postoperatively. Taste buds were present in three of the four papillae examined 30 weeks after nodose ganglion transplantation (Fig. 4D). In one papilla 12 buds were found while the others had 14 and 19; these are far fewer than the hundreds of buds normally present in the papilla (6). The regenerated around the transplanted nerve trunks which consists of large-nucleated cells (? macrophages) (arrow, m), neutrophilic leucocytes (arrow, n), and small lymphocytes (arrow, L). B. Glossopharyngeal nerve end of the arterial graft anastomosis site. Many nerve fibers from the nodose ganglion have grown into the distal glossopharyngeal nerve trunk (arrow, gt) while other vagal fibers (unlabeled arrows) have regenerated around it.
ZALEWSKI
X70. -4. Normal papilla. Numerous taste FIG. 4. Adenosine triphosphatase activity. buds are present in the epithelium of the trench walls. B. Five-day glossopharyngeal nerve denervated papilla. The number of taste buds is reduced, and those buds present are in various stages of degeneration. C. Ten-day glossopharyngeal nerve denervated papilla. All taste buds have disappeared. D. Thirty-week homologous nodose ganglion transplant reinnervated papilla. Regenerated taste buds are randomly located in the trench walls.
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FIG. 5. A-G. Adenosine triphosphatase activity. A. Normal trench wall. Normal buds extend through the entire width of the epithelium, and all the taste cells exhibit intense enzyme activity. X 300. El. Five-day glossopharyngeal nerve denervated trench wall. Degenerating buds have moved away from the basement membrane toward the epithelial surface and they are smaller than normal and contain fewer taste cells. X300. C. Thirty-week glossopharyngeal nerve denervated trench wall. No taste buds are present, and the trench wall epithelium is atrophied. X300. D. Thirty-week homoogous nodose transplant reinnervated trench wall. The regenerated bud, like normal ones, extends through the entire width of the epithelium and has numerous taste ce!ls with intense adenosine triphophatase activity. X 300. E-G. Thirty-week homologous nodose ganglion transplant reinnervated and subsequently 5-day denervated papilla (E) ( X 70) and trench walls (F and G) ( X 300). Two degenerating buds are seen in the epithelium of the trench walls. H-K. Cholinesterase activity. X300. An intense nerve fiber cholinesterase reaction is present immediately beneath the epithlium where a normal glossopharyngeal innervated bud is located (arrow) (H). Enzyme activity is also present beneath the ganglion transplant reinnervated bud (arrow) (I), but it disappears after removing the ganglion within 5 days from beneath the degenerating buds (arrow) (J and K).
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buds were randomly located in the trench wall epithelium, and each estended the full width of the epithelium with taste cells showing intense adenosine triphosphatase activity (Fig. SD). A moderate cholinesterase reaction was present beneath the epithelium where the regenerated buds were located (Fig. 51). Only occasional tracs of cholinesterase activity were seen near the epithelium of the one papilla in which no taste buds were found. Degenerating buds were found in three of the five papilla studied 5 days after denervating the ganglion transplant reinnervated papilla (Fig. 5E). These buds, as in the previously described 5-day glossopharyngeal denervated papilla, were small, had few cells, and were present only near the surface of the trench wall (Fig. 5F and G). No cholinesterase activity was seen beneath these clegenerating buds (Fig. 5J and K). The remaining two papillae lacked taste buds, and it is therefore not certain whether buds were present prior to the denervation or whether all buds had disappeared within the 5-day postoperative interval.
Cellular Response to the Hovlzologously Transplanted Nodose Ganglia and Nerve Fibers. A mild cellular reaction of large-nucleated cells, neutrophilic leucocytes, and small lymphocytes was present around but not in the transplanted nodose ganglia (Fig. IB) and its nerve trunks (Fig. 3A). The latter two cell types were identified because they gave the same type of cholinesterase stain when they were compared to peripheral blood. No such cellular reaction was seen in the distal glossopharyngeal nerve trunk into which the vagal nerve fibers had regenerated or in the papilla.
Observations Concerning Vascularization of the Transplanted Nodose Ga,ngZion. No direct vascular tracer studies of the transplanted ganglia were performed, but in the ganglia some neutrophilic leucocytes were found. These cells were clearly identified because they are the only normal leucocytes that stain for alkaline pbosphatase activity (16, 18). These leucocytes were few in number and generally found near capillary endothelium (which also stains intensely for alkaline phosphatase). It seems unlikely that these cells represent donor rather than host leucocytes because neutrophilic leucocytes have a lo-14-day life-span, and any donor leucocytes which may have been present in the ganglion at the time of transplantation should have disappeared after 30 weeks. Discussion
The histological studies show that some neurons in the homologously transplanted vagal nodose ganglion survived and that they exhibited a morphology and enzyme activity and distribution similar to that of normal nodose neurons. Other studies of transplanted sensory ganglia have also indicated that some neurons can survive homologous transplantation and,
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after undergoing an initial chromatolytic reaction, return to a normal morphological appearance ( 13, 15). However, since transplantation in these studies was made into the brain (13, 15) there was little opportunity to determine if nerve fiber growth or functional reinnervation occurred. In the present study, the neurons not only survived, but some of them regenerated their nerve fibers to the tongue and caused taste bud regeneration. The only other system where transplantd ganglion neurons have been demonstrated to perform a trophic function concerns studies on limb regeneration in the amphibian. In this study it was shown that homologous transplants of spinal ganglia could sustain blastemal growth during the critical period when growth was dependent on the nerve (9). Furthermore, a recent study has demonstrated the chemical nature of this neurotrophic influence by showing that infusions of nerve homogenates will stimulate protein synthesis in the regenerating blastema (10). However, it is uncertain whether the neurons in the transplanted ganglion maintained blastema growth (9) in a physiological manner or whether dead or dying neurons merely liberated a biologically active chemical substance into the blastema for either process seemingly could function as an infused nerve homogenate. This question might be resolved by an experiment in which the ganglia are transplanted several months prior to amputation of the limb; if the limb regenerated it would show that the neurons can exert their tropbic function even after prolonged periods of transplantation. Since taste bud cells are known to undergo a physiological turnover (2)) it appears certain from the results of the present study that the transplanted neurons are performing a trophic function by a physiological mechanism. The fact that the transplanted neurons retained their trophic function does not mean that all aspects of neuronal or taste bud metabolism are normal. For example, impulse conduction, other aspects of neuron morphology or histochemistry. or taste responses might be abnormal, especially since it is highly probable that not all neurons grew out fibers that reinnervated appropriate end organs. Consequently these parameters must be further investigated before the transplanted neurons or regenerated taste buds can be considered fully normal. Neuronal metabolism, in fact, can be altered without the loss of trophic function for it has been shown that chromatolyzed gustatory neurons still can support taste buds (20). The presence of degenerating buds 5 days after transection of the regenerated nodose nerve fibers further demonstrates that the transplanted neurons had indeed performed their trophic function, for in all regards, the degenerating buds resembled those seen after acute transection of the normally glossopharyngeal innervated papilla. Inasmuch as the previous studies of homogously transplanted ganglia
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(9, 13, 15) were performed before the basic principles of immunological tissue tolerance were known, it is understandable why no mention was made of cellular reaction to the transplants. The cellular response to the ganglion transplant was, however, an important immunoiogical question that was asked and described in the present study. It should be understood that in order to elicit an immune response the transplanted tissue must be sufficiently antigenic (qualitatively and quantitatively‘) to elicit an immune response, the antigen must enter the lymphatic tissue and stimulate antibody production to it, the antibodies must have a vascular means of getting to the source of the antigen, and the antibody must have the opportunity to freely interact with the antigenic tissue (11). Brain homografts (e.g.. cortex, cerebellum, corpus callosum) have heen reported to induce sensitization inasmuch as skin grafts performed several weeks later are rejected at an accelerated rate (1). However, in this study, no neurons survived transplantation so that antigenicity of the brain tissue was attributed to surviving glial tissue. Before neurons can be studied for antigenicity, transplantation conditions must be designed to promote vascularization so that anoxia can be eliminated as a cause of neuron death. This has been accomplished in homologous heart transplantation studies in which vascular anastomosis is readily accomplished. Early time studies of neurons in intrinsic heart autonomic ganglia of dogs showed histologically normal neurons that were surrounded by a cellular reaction similar to that which occurred around the heart muscle fibers at the time of cardiac failure (14). It was suggested, as in the previous study (1). that the nonneuronal elements of nerve tissue (i.e., neuroglia, myelin. neurilemma cells) were the strongly antigenic component. In the present study neurons survived, regenerated their nerve fibers, and performed a trophic function despite the presence of a cellular response. The total numbr of neurons surviving chronic homologous transplantation did not appear different from the number seen after antologous transplantation in ZGZV (21). It seems therefore that neuron death in the homologously transplanted ganglia was due to some transplantation shock rather than an immune response. A notable difference between the cardiac and ganglion transplants is that in the former, a cellular rejection reaction occurred around the neurons in the ganglia. In the nodose ganglion it was present around the ganglion capsule and connective tissue of the vagal nerve trunks but not in the ganglion core where the neurons are located nor in the fascicles containing the nerve fibers. Further investigations are needed to evaluate the antigenicity of neurons. These studies must employ highly imbred but antigenically incompatible animals as donors and hosts rather than the random-bred unmatched animals used in the present study. First- and second-set rejection studies must
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also be included to determine whether neurons possessno immunologically competent antigen, a unique antigen, or a common tissue antigen. It is clear though that some neurons in homologous ganglion transplants can survive and trophically function for prolonged periods. References 1. BARNES, A. D. 1969. The immunogenicity of brain, ovary, and skin allografts in mice. Transfilantation 8 : 379-382. 2. BEIDLER, L. M.. and R. L. SMALLXXAN. 1965. Renewal of cells within taste buds. J. Cell Biol. 27 : 263-272. 3. BURSTONE, M. S. 1961. Histochemical demonstration of phosphatases in frozen sections with Naphtha1 AS-phosphates. J. Histochem. Cytockem. 9 : 146156. 4. GOLDBERG, A. F., and T. BARKA. 1962. Acid phosphatase activity in human blood cells. Nature London 195 : 297. 5. G~MORI, G. 1952. “Microscopic Histochemistry : Principles and Practice.” Univer. of Chicago Press, Chicago, Ill. 6. GUTH, L. 1957. The effects of glossopharyngeal nerve transection on the circumvallate papilla of the rat. Anat. Rec. 129 : 715-731. 7. GUTH, L. 1969. Trophic effects of vertebrate neurons. Neurosci. Res. Program Bull. 7 : l-73. 8. HENDERSON, J. R. 1967. The use of silver for intensifying sulfide deposits in the cholinesterase technique. Stain Tecknol. 42 : 101-102. 9. KAMRIN, A. A., and M. SINGER. 1959. The growth influence of spinal ganglia implanted into the denervated forelimb regenerate of the newt, Triturus. J. Morjkol. 225 : 82M27. 10. LIEBOWITZ. P., and M. SINGER. 1970. Neurotrophic control of protein synthesis in the regenerating limb of the newt, Triturus. Nature London 225: 824-827. 11. MEDAWAR, P. B. 1948. Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Brit. J. Exp. Patkol. 29 : 5869. 12. PEARSE, A. G. E. 1961. Methods (#2) for succinate dehydrogenase using MTT, p. 910. In “Histochemistry, Theoretical, and Applied,” 2nd ed. Little, Brown, Boston. 13. RANSON, S. W. 1914. Transplantation of the spinal ganglion with observations on the significance of the complex types of spinal ganglion cells. J. Cofnp. Neural. 24 : 547-558. 14. SHARDEY, G. C., E. COOPER, and W. G. R. M. DE BOER. 1970. Differential rejection of neurones and neuroglia in canine cardiac allografts. Nature London 228: 69-71. 15. TIDD, C. W. 1932. The transplantation of spinal ganglia in the white rat. A study of the morphological changes in surviving cells. J. Co”zp. Neural. 55: 531-543. 16. TITTOBELLO, A., and A. AGOSTONI. 1967. Leucoycyte alkaline phosphatase: A quantitative and qualitative study in normal and leukemic cells. Enzyvmol. Biol. C&z. 8 : 413420. 17. WACHSTEIN, M., and E. MEISEI.. 1957. Histochemistry of hepatic phosphatase at a physiologic pH. Amer. J. Clin. Pathol. 27 : 13-23. 18. ZALEWSKI, A. A. 1969a. Combined effects of testosterone and motor, sensory, or gustatory reinnervation on the regeneration of taste buds in the rat. E.rp. Neural. 24 : 285-297.
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A. A. 1969b. Regeneration of taste buds after reinnervation by perior central fibers of vagal ganglia. Exp. Neural. 25 : 429-437. 20. ZALEWSKI, A. A. 1970. Continuous trophic influence of chromatolyzed gustatory neurons on taste buds. Aeat. Rec. 167 : 165-173. 21. ZALEWSKI, A. ‘4. 1970. Trophic influence of irk Cm transplanted sensory neurons on taste buds. Exb. h’clruol. 29: 462-467. 19.
Z.~LE~VSKI,
pheral