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Trophic function of neurons in transplanted neonatal ganglia
ESPEHIMENTAL
45, 189-193
XEUHOLt~GY
(
1974)
RESEARCH Trophic
NOTE
Function of Neurons in Transplanted Neonatal Ganglia ANDRE\V A. ZALEWSKI 1
Laborafoq Strokr,
of
N‘~lrrochc~~ti~~fr~I, Natioriai Institute Nutional Imtitzltcs of Health, U. Edrtcario)z. CIFI~ 11 7clftrrc, Bethesda,
of S.
Nczrrological
.Vfnr~~l~~~rti
Lhruscs
of
Dcpartntcut
awl
Hmlth.
.?0014
Previous studies demonstrated that some neurons in autografts and homografts of sensory ganglia survive for prolonged periods after transplantation and that they can regenerate nerve fibers into tongue tissue and cause the regeneration of taste buds (G-9). Since these results were obtained with grafts taken from animals that were between 70-150 clays old, it seemed pertinent to determine whether neurons in grafts donated by younger animals (i.e., 22-day-old and neonatal ) would similarly survive and function. While the total number of neurons is comparable in ganglia from young and adult animals, they differ significantly in their state of maturation (3) and in their reaction to injury (4). For this reason. the age of the grafted neurons might determine their ability to survive the trauma of transplantation and to mature. regenerate nerve fibers, and functionally reinnervate en&organs. The present study was, therefore, performed to determine at what age neurons could survive transplantation and whethel these surviving neurons could reinnervate their own comparably aged or adult tissue. An inbred strain of Fischer rats was used. The nodose ganglion and the tongue’s vallate papilla were removed from neonatal (less than 20-hr-old ) or 22-day-old rats and transplanted to the anterior chamber of the eye of other young adult (70- to SO-day-old) host rats. Neonatal and 22-day-old ganglia were selected for study because neurons in neonatal ganglia are un1 Mr. George Creswell throughout
provided excellent surgical
this study. 189
Copyright All rights
Q 1974 by Academic Press, of reproduction in any form
and
histological
assistance
differentiated
while
neurons
of Z-day-old
rats have just matured
(3).
The
papilla was placed over the peripheral end of the ganglion so that nerve fibers from any surviving neurons would have the opportunity to grow into it. By this procedure I could test the functional ability of surviving neurons to exert a neurotrophic influence and thereby induce the regeneration and maintenance of taste buds in the papilla (2). As a control procedure some tongue grafts were transplanted to the eye without the ganglia. The grafts were removed at various times after transplantation and S-pm frozen sections of them stained with PAS-hematoxylin, adenosine triphosphatase (ATPase) (1)) and cholinesterase (5) techniques. In addition, normal neonatal and 22-day-old nodose ganglia and normal vallate papillae were examined.
Swvival of Nez~ons. Six grafts of combined neonatal ganglia and neonatal tongue and six grafts of combined 22-day-old ganglia and tongue were studied 35-70 days after transplantation. Neurons were present in all transplanted ganglia (Fig. 1B) but their number was reduced when compared to normal ganglia (Fig. IA). There was some variability in the number of neurons which survived in each transplanted ganglion but over-all the number of neurons present in transplanted neonatal ganglia was similar to that present in transplanted 22-day-old ganglia. Mat14ration of Nez~ons. Initially the neonatal neurons were fairly uniform in size and their nerve fibers were essentially unmyelinated. They differentiated into large- and small-sized cells and their nerve fibers became myelinated while in the anterior chamber of the eye. Thus, some neonatal and 22-day-old neurons not only survived the trauma of transplantation but, moreover, the neonatal neurons continued to differentiate. Taste Buds. Examination of the tongue grafts that were combined with these ganglia showed that all six of the 22-day-old tongue grafts later contained regenerated taste buds. Counts of taste buds in each papilla showed that 4, 20, 24, 29, 30, and 47 were present. Three of the six neonatal tongue grafts later had 8, 24, and 28 regenerated buds. Taste buds were readily identified becausethe taste cells composing them stained intensely for adenosine triphosphatase activity. In a normal papilla the buds were symmetrically arranged in the epithelium of the inner and outer trench walls (Fig. 2A). However, in the transplanted papillae, buds were found randomly distributed in the trench walls (Fig. 2C, D) and even in the epithelium on the top of the papillae (Fig. 2C). The regenerated buds, like normal ones, extended through the width of the epithelium (Fig. 2E), and under each bud the cholinesterase activity of nerve fibers could be demonstrated (Fig. 2F). Taste buds were absent from all three neonatal and all three 22-day-old papillae that had been transplanted without nodose ganglia (Fig. 2B). Since neurons in both neonatal and 22-day-old ganglia
FIG. 1. -4 and B. Pr\S-hematoxylin stain ( X 1.50 ). A. Normal nodose ganglion from a 65day-old rat. Many neurons (arrow N) and myclinatetl nerve fihcrs (unlabeled arrOW4) are present. B. ~odose gdilg~ioll ta!ien from a neonatal rat and transplanted for 65 days to the anterior chamber of the eye. iYeurons (arro~v N\‘) are present hut in reduced numbers and myelinatetl nerve fibers (unlabeled arrows) which are essentially absent in a neonatal ganglion have appeared. The myelin of nerve fihers stains red kvith the P;IS stain and is readily distinguished from nuclei which stain hlue with hematoxylin. .Arro\\ IR of B shows some hoat iris tissue which became attached to thr transplanted ganglion.
survived after transplantation order to determine whether Consequently. six neonatal
it was decided to use only neonatal ganglia in younger neurons could reinnervate adult tissue. ganglia were combined with six papillae that
192
ANDREW
A. ZALEWSKI
FIG. 2. A-E. ATPase stain; F, cholinesterase-hematoxylin stain. A. Normal papilla (X 70). Taste buds are found in the epithelium of the trench walls (arrows) but not in the epithelium covering the top of the papilla (arrow TP). B. Papilla transplanted without ganglion (X 70). No taste buds are present in the trench walls (arrows). C-F. Papilla combined with ganglia. Regenerated taste buds are randomly distributed in the trench walls (arrows of C, D) (X 70) and some are even present in the epithelium on the top of the papilla (arrow TP of C). The regenerated buds extended through the width of the epithelium (E) (X 300), and under each bud nerve fiber cholinesterase activity (arrows of F) (X 300) was demonstrated.
XEONATAL
193
GANGLIA
were takeli ftYJll1 tlollurs over 1SO days old. Five of these papillw wl)sv quently contained 6, 18. 2i. 32. and 36 regenerated taste buds. The results of the present and past studies (6-9) demonstrate that neurons taken from neonatal, weanling, (2.2~day-old), or adult rats can be successfully transplanted and that they can establish a functional innervation as revealed by their ability to induce taste bud regeneration. Furthermore, since the present studies also show that transplanted neonatal neurons can reinnervate adult tissue it is now possible to use a donor of any age as a source of neuronal grafts. Finally. it can be anticipated that homografts of neonatal ganglia can be used similarly to that described above since it has already been demonstrated that the immunological rejection of neurons that occurs in homografts of adult ganglia can be prevented (9) and that these grafted neurons are functional 18 ). KEFEKENCES 1. GOMORI,
of 2. GUTH,
G. 1952. “Microscopic Histochemistry Chicago Press, Chicago, Illinois. L. 1969. Trophic effects of vertebrate
: Principles neurons.
and
Practice.”
Univ.
h’crtrosci. Kc-s. Pro,urtrr/~
131111. 7 : l-73. 3.
M., and M. WOL~IAX. 1970. Correlative study on the maturation of sensory ganglion 100-108. BELLE. A. 1964. Critical periods of neuronal
KALINA,
4. LA
histochemical cells in the maturation.
and rat,
morphological
Histocltcmir
22:
firnirl
Krs.
Pro!/?‘.
9: 93-96. and E. MEISEL. 1957. Histochemistry of hepatic phosphatase at a pH. .Jrrrrv. J. C/ix. P~thol. 27 : 13-23. 6. ZALE\VSKI, A. A. 1971. The effect of Ag-B locus compatibility and incompatibility on neuron survival in transplanted sensory ganglia in rats. E.rp. NrUrol. 33:
5. WACHSTEIN,
M.,
physiologic
576-583. 7.
A. A. 1972. Trophic function of homograftetl neurons oi .\g-B-historats. TrcJrls~lorlttrticllr 14 : 618-623. 8. ZALE\VSKI, .4. .4., and W. K. SILVERS. 1973. Trophic iunction of neurons in homografts of ganglia in immunologically tolerant rats. Krp. KcI(I.o/. 41 : 777-781. 9. ZALE~SGI, A. .4.. and W. K. SILVEKS. 1974. Survival of neurons in homografts of ganglia in adult rats neonatally treated with hone marrow or lymph node cells. Amt. KC-C. 178 : 243-2.52. ZALEWSKI,
compatible
45, 189-193
XEUHOLt~GY
(
1974)
RESEARCH Trophic
NOTE
Function of Neurons in Transplanted Neonatal Ganglia ANDRE\V A. ZALEWSKI 1
Laborafoq Strokr,
of
N‘~lrrochc~~ti~~fr~I, Natioriai Institute Nutional Imtitzltcs of Health, U. Edrtcario)z. CIFI~ 11 7clftrrc, Bethesda,
of S.
Nczrrological
.Vfnr~~l~~~rti
Lhruscs
of
Dcpartntcut
awl
Hmlth.
.?0014
Previous studies demonstrated that some neurons in autografts and homografts of sensory ganglia survive for prolonged periods after transplantation and that they can regenerate nerve fibers into tongue tissue and cause the regeneration of taste buds (G-9). Since these results were obtained with grafts taken from animals that were between 70-150 clays old, it seemed pertinent to determine whether neurons in grafts donated by younger animals (i.e., 22-day-old and neonatal ) would similarly survive and function. While the total number of neurons is comparable in ganglia from young and adult animals, they differ significantly in their state of maturation (3) and in their reaction to injury (4). For this reason. the age of the grafted neurons might determine their ability to survive the trauma of transplantation and to mature. regenerate nerve fibers, and functionally reinnervate en&organs. The present study was, therefore, performed to determine at what age neurons could survive transplantation and whethel these surviving neurons could reinnervate their own comparably aged or adult tissue. An inbred strain of Fischer rats was used. The nodose ganglion and the tongue’s vallate papilla were removed from neonatal (less than 20-hr-old ) or 22-day-old rats and transplanted to the anterior chamber of the eye of other young adult (70- to SO-day-old) host rats. Neonatal and 22-day-old ganglia were selected for study because neurons in neonatal ganglia are un1 Mr. George Creswell throughout
provided excellent surgical
this study. 189
Copyright All rights
Q 1974 by Academic Press, of reproduction in any form
and
histological
assistance
differentiated
while
neurons
of Z-day-old
rats have just matured
(3).
The
papilla was placed over the peripheral end of the ganglion so that nerve fibers from any surviving neurons would have the opportunity to grow into it. By this procedure I could test the functional ability of surviving neurons to exert a neurotrophic influence and thereby induce the regeneration and maintenance of taste buds in the papilla (2). As a control procedure some tongue grafts were transplanted to the eye without the ganglia. The grafts were removed at various times after transplantation and S-pm frozen sections of them stained with PAS-hematoxylin, adenosine triphosphatase (ATPase) (1)) and cholinesterase (5) techniques. In addition, normal neonatal and 22-day-old nodose ganglia and normal vallate papillae were examined.
Swvival of Nez~ons. Six grafts of combined neonatal ganglia and neonatal tongue and six grafts of combined 22-day-old ganglia and tongue were studied 35-70 days after transplantation. Neurons were present in all transplanted ganglia (Fig. 1B) but their number was reduced when compared to normal ganglia (Fig. IA). There was some variability in the number of neurons which survived in each transplanted ganglion but over-all the number of neurons present in transplanted neonatal ganglia was similar to that present in transplanted 22-day-old ganglia. Mat14ration of Nez~ons. Initially the neonatal neurons were fairly uniform in size and their nerve fibers were essentially unmyelinated. They differentiated into large- and small-sized cells and their nerve fibers became myelinated while in the anterior chamber of the eye. Thus, some neonatal and 22-day-old neurons not only survived the trauma of transplantation but, moreover, the neonatal neurons continued to differentiate. Taste Buds. Examination of the tongue grafts that were combined with these ganglia showed that all six of the 22-day-old tongue grafts later contained regenerated taste buds. Counts of taste buds in each papilla showed that 4, 20, 24, 29, 30, and 47 were present. Three of the six neonatal tongue grafts later had 8, 24, and 28 regenerated buds. Taste buds were readily identified becausethe taste cells composing them stained intensely for adenosine triphosphatase activity. In a normal papilla the buds were symmetrically arranged in the epithelium of the inner and outer trench walls (Fig. 2A). However, in the transplanted papillae, buds were found randomly distributed in the trench walls (Fig. 2C, D) and even in the epithelium on the top of the papillae (Fig. 2C). The regenerated buds, like normal ones, extended through the width of the epithelium (Fig. 2E), and under each bud the cholinesterase activity of nerve fibers could be demonstrated (Fig. 2F). Taste buds were absent from all three neonatal and all three 22-day-old papillae that had been transplanted without nodose ganglia (Fig. 2B). Since neurons in both neonatal and 22-day-old ganglia
FIG. 1. -4 and B. Pr\S-hematoxylin stain ( X 1.50 ). A. Normal nodose ganglion from a 65day-old rat. Many neurons (arrow N) and myclinatetl nerve fihcrs (unlabeled arrOW4) are present. B. ~odose gdilg~ioll ta!ien from a neonatal rat and transplanted for 65 days to the anterior chamber of the eye. iYeurons (arro~v N\‘) are present hut in reduced numbers and myelinatetl nerve fibers (unlabeled arrows) which are essentially absent in a neonatal ganglion have appeared. The myelin of nerve fihers stains red kvith the P;IS stain and is readily distinguished from nuclei which stain hlue with hematoxylin. .Arro\\ IR of B shows some hoat iris tissue which became attached to thr transplanted ganglion.
survived after transplantation order to determine whether Consequently. six neonatal
it was decided to use only neonatal ganglia in younger neurons could reinnervate adult tissue. ganglia were combined with six papillae that
192
ANDREW
A. ZALEWSKI
FIG. 2. A-E. ATPase stain; F, cholinesterase-hematoxylin stain. A. Normal papilla (X 70). Taste buds are found in the epithelium of the trench walls (arrows) but not in the epithelium covering the top of the papilla (arrow TP). B. Papilla transplanted without ganglion (X 70). No taste buds are present in the trench walls (arrows). C-F. Papilla combined with ganglia. Regenerated taste buds are randomly distributed in the trench walls (arrows of C, D) (X 70) and some are even present in the epithelium on the top of the papilla (arrow TP of C). The regenerated buds extended through the width of the epithelium (E) (X 300), and under each bud nerve fiber cholinesterase activity (arrows of F) (X 300) was demonstrated.
XEONATAL
193
GANGLIA
were takeli ftYJll1 tlollurs over 1SO days old. Five of these papillw wl)sv quently contained 6, 18. 2i. 32. and 36 regenerated taste buds. The results of the present and past studies (6-9) demonstrate that neurons taken from neonatal, weanling, (2.2~day-old), or adult rats can be successfully transplanted and that they can establish a functional innervation as revealed by their ability to induce taste bud regeneration. Furthermore, since the present studies also show that transplanted neonatal neurons can reinnervate adult tissue it is now possible to use a donor of any age as a source of neuronal grafts. Finally. it can be anticipated that homografts of neonatal ganglia can be used similarly to that described above since it has already been demonstrated that the immunological rejection of neurons that occurs in homografts of adult ganglia can be prevented (9) and that these grafted neurons are functional 18 ). KEFEKENCES 1. GOMORI,
of 2. GUTH,
G. 1952. “Microscopic Histochemistry Chicago Press, Chicago, Illinois. L. 1969. Trophic effects of vertebrate
: Principles neurons.
and
Practice.”
Univ.
h’crtrosci. Kc-s. Pro,urtrr/~
131111. 7 : l-73. 3.
M., and M. WOL~IAX. 1970. Correlative study on the maturation of sensory ganglion 100-108. BELLE. A. 1964. Critical periods of neuronal
KALINA,
4. LA
histochemical cells in the maturation.
and rat,
morphological
Histocltcmir
22:
firnirl
Krs.
Pro!/?‘.
9: 93-96. and E. MEISEL. 1957. Histochemistry of hepatic phosphatase at a pH. .Jrrrrv. J. C/ix. P~thol. 27 : 13-23. 6. ZALE\VSKI, A. A. 1971. The effect of Ag-B locus compatibility and incompatibility on neuron survival in transplanted sensory ganglia in rats. E.rp. NrUrol. 33:
5. WACHSTEIN,
M.,
physiologic
576-583. 7.
A. A. 1972. Trophic function of homograftetl neurons oi .\g-B-historats. TrcJrls~lorlttrticllr 14 : 618-623. 8. ZALE\VSKI, .4. .4., and W. K. SILVERS. 1973. Trophic iunction of neurons in homografts of ganglia in immunologically tolerant rats. Krp. KcI(I.o/. 41 : 777-781. 9. ZALE~SGI, A. .4.. and W. K. SILVEKS. 1974. Survival of neurons in homografts of ganglia in adult rats neonatally treated with hone marrow or lymph node cells. Amt. KC-C. 178 : 243-2.52. ZALEWSKI,
compatible