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Survival, regeneration, and trophic function of neurons in 1-year transplants of sensory ganglia
EXPERIMENTAL
NEUROLOGY
68,
390-394
(1980)
RESEARCH Survival,
NOTE
Regeneration, and Trophic Function of Neurons in l-Year Transplants of Sensory Ganglia ANDREW
A. ZALEWSKI
Laboratory of Neurochemistry, National Institute Communicative Disorders and Stroke. National Bethesda, Maryland 20205 Received
August
14. 1979; retsision
receir,ed
of Neurologicul and Institutes of Health,
November
6. 1979
Previous studies showed that some neurons survive in grafts of ganglia placed into the anterior chamber of the eye and that these neurons can regenerate their nerve fibers into cotransplanted tongue grafts where they induce the formation of taste buds (8- 11). In the present study ganglia were transplanted into eyes but no tongue grafts were added until 1 year later. The purpose of this experiment was to determine whether or not trophic sensory neurons, that is, the neurons responsible for taste bud development and maintenance, would survive after long-term isolation from their end-organs and still maintain their ability to regenerate and induce taste buds after exposure to a tongue graft. Isogenic strains of Brown Norway male rats, weighing 200 to 250 g, were used. An isograft of the sensory vagal nodose ganglion, which consisted of the ganglion and 4 to 6 mm of attached peripheral nerve trunk, was transplanted into the anterior chamber (i.e., the fluid-filled space between the cornea and iris) of the eye of the host rat. One year later an isograft of tongue tissue (taken from a rat different from the one that donated the ganglion) that contained the taste bud-bearing vallate papilla was added to the eye of 10 rats which had a graft of ganglion. The vallate papilla in the graft contained its normal component of taste buds and nerve fibers (i.e., it was not denervated prior to grafting). The tongue graft was adjusted in the eye so that the connective tissue surface of it rested on top of the end of the peripheral nerve trunk of the ganglion. As a control procedure, a 390 0014-4886/80/050390-05$02.00/O
GANGLIA
TRANSPLANTS
391
tongue graft was transplanted alone into the other eye of six of the rats which had combined ganglion-tongue grafts. The techniques of obtaining, trimming, and inserting the grafts were the same as previously reported (11). All grafts were removed 35 days after addition of tongue tissue to the eyes. The ganglia and tongue grafts were separated and each graft was placed between different slabs of skeletal muscle in preparation for freezing in liquid nitrogen. Eight-micrometer-thick frozen cross sections of the grafts were prepared and some stained by periodic acidSchiff hematoxylin (PAS-hematoxylin), adenosine triphosphatase
FIG. I. Normal (A) and transplanted (B) nodose ganglia. Many neurons (one indicated by long arrow) and regions of myelinated nerve fibers (some indicated by short arrow) are present in a normal nodose ganglion (A) whereas reduced numbers of neurons and myelinated nerve fibers are present in a transplanted (B) ganglion. The dark tissue labeled Ir in B is pigmented host iris to which the transplanted ganglion attaches while residing in the eye. PAS-Hematoxylin stain, x 1 IO.
392
ANDREW
A. ZALEWSKI
(ATPase) (7), or cholinesterase (2) procedures. Normal nodose ganglia and vallate papillae were similarly examined. The presence, number, and distribution of neurons and myelinated nerve fibers in a normal nodose ganglion are shown in Fig. 1A. At the time the tongue grafts were added to the eyes, all ganglia were visible and although they were somewhat smaller than normal there was no progressive reduction in size throughout their period of transplantation. Histological examination of the transplanted ganglia revealed that each contained neurons but these were present in reduced numbers (Fig. 1B) compared to a normal ganglion (Fig. 1A). Each transplanted ganglion, however, seemed to have similar numbers of surviving neurons. Many of the surviving neurons exhibited reduced Nissl substance and a displaced nucleus. Myelin staining was demonstrable around nerve fibers in the ganglia but no cellular inflammatory reaction was noted. It is noteworthy that l-year grafts of ganglia had similar numbers of surviving neurons compared to neurons in nodose ganglia grafts that were studied at shorter time periods of previous experiments (8-10). All tongue grafts that were combined with grafts of ganglia had regenerated taste buds (Figs. 2B-D). For comparison purposes, a normal vallate is illustrated in Fig. 2A where it is seen that taste buds are present only in the epithelium of the trench walls of the papilla. In contrast, the regenerated taste buds in tongue grafts were randomly situated in the trench walls (Fig. 2B) and in four grafts some buds were found even in the epithelium on the top surface of the papilla (Fig. 2C). The number of regenerated buds per papilla ranged from 14 to 42. A cholinesterase reaction, indicative of the presence of nerve fibers (9), was always found beneath the epithelium which contained the regenerated taste buds (Fig. 2D). No taste buds were found in any of the six tongue grafts that were transplanted alone (i.e., without ganglion) to eyes (Fig. 2E). The present results demonstrate that sensory neurons can survive longterm transplantation in isolation from their normal tissue. This result contrasts with sympathetic neurons which seem to decrease in number following long transplant periods (4). More importantly, the present study shows that neurons can, after chronic isolation, reinitiate the regeneration of their nerve fibers if they are reexposed to their appropriate tissue. This observation may have implications for reinitiating the regeneration of the injured central nervous system because apparently some nerve fiber growth-stimulating factor(s) is present in grafted tissue. Indeed, Seiger and Olson (5) and Bjorklund and Stenevi (1) have already shown that central nervous system neurons can respond by regeneration if exposed to appropriate tissue grafts. Seiger and Olson (6) also have data which indicate that the growth-promoting factor(s) in grafted tissue is related to
GANGLIA
TRANSPLANTS
393
FIG. 2. Normal vallate papilla (A) and papillae combined with ganglia (B-D) or transplanted alone (E) to eyes. Taste buds are present in the epithelium of the trench walls (arrows) and not in the epithelium on the top surface (indicated by lettering Tp) of a normal papilla (A). Randomly regenerated taste buds are present in the epithelium of the trench walls (arrow of B) and top surface (region bounded by arrows in C) of papillae that were combined with ganglia. A cholinesterase reaction in nerve tissue (arrow of D) is seen beneath the epithelium which supports a regenerated taste bud. No taste buds are present in a papilla transplanted alone to the eye (E). A-C, E. ATPase stain, x70: D. cholinesterase hematoxylin stain, x300.
innervation because grafts of chronically denervated tissue are a poor inducer of axonal outgrowth. It is possible, therefore, that the repair of central nervous system tissue might be accomplished by using a nerve tissue graft (3) and growth factors obtained from grafted tissue. In this scheme, a nerve graft would provide a bridge through which growth factor-stimulated axons might regenerate. REFERENCES I, BJORKLUND. A., AND U. STENEVI. 1979. Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system. Phxsiol. Rev. 59: 62- 100. 2. GOMORI. G. 1952. Microscopic Histochrrnistr~: Principlrs and Practice. Univ. of Chicago Press, Chicago.
394
ANDREW
A. ZALEWSKI
3. KAO, C. C., L. W. CHANG, AND J. M. B. BLOODWORTH, JR. 1977. The mechanism of spinal cord cavitation following spinal cord transection. Part 3. Delayed grafting with and without spinal retransection. J. Neurosurg. 46: 757-766. 4. OLSON, L., AND T. MALMFORS. 1970. Growth characteristics ofadrenergic nerves in the adult rat. Acts Physiol. Stand. Suppl. 348: l-112. 5. SEIGER. A., AND L. OLSON. 1977a. Growth of locus coeruleus neurons in oculo independent of simultaneously present adrenergic and cholinergic nerves in the iris. Med. Biol.
55: 209-223.
6. SEIGER, A., AND OLSON, L. 1977b. Reinitiation of directed nerve fiber growth in central monoamine neurons after intraocular maturation. Exp. Brain Res. 29: 15-44. 7. WACHSTEIN, M., AND E. MEISEL. 1957. Histochemistry of hepatic phosphatase at a physiological pH. Am. J. Clin. Pathol. 27: 13-23. 8. ZALEWSKI, A. A., AND W. K. SILVERS. 1973. Trophic function of neurons in homografts of ganglia in immunologically tolerant rats. &I. Neural. 41: 777-781. 9. ZALEWSKI, A. A. 1976. The neural induction of taste buds in the salivary ducts of the lingual gland of Von Ebner. Exp. Neural. 52: 565-580. 10. ZALEWSKI, A. A., AND W. K. SILVERS. 1977. The long-term fate of neurons in allografts of ganglia in Ag-B-compatible normal and immunologically tolerant rats. J. Neurobiol.
8: 207-215.
11. ZALEWSKI, A. A. 1979. The distribution of alkaline phosphatase activity in normal and cross-species regenerated rat and mouse taste buds. Anat. Rec. 194: 283-292.
NEUROLOGY
68,
390-394
(1980)
RESEARCH Survival,
NOTE
Regeneration, and Trophic Function of Neurons in l-Year Transplants of Sensory Ganglia ANDREW
A. ZALEWSKI
Laboratory of Neurochemistry, National Institute Communicative Disorders and Stroke. National Bethesda, Maryland 20205 Received
August
14. 1979; retsision
receir,ed
of Neurologicul and Institutes of Health,
November
6. 1979
Previous studies showed that some neurons survive in grafts of ganglia placed into the anterior chamber of the eye and that these neurons can regenerate their nerve fibers into cotransplanted tongue grafts where they induce the formation of taste buds (8- 11). In the present study ganglia were transplanted into eyes but no tongue grafts were added until 1 year later. The purpose of this experiment was to determine whether or not trophic sensory neurons, that is, the neurons responsible for taste bud development and maintenance, would survive after long-term isolation from their end-organs and still maintain their ability to regenerate and induce taste buds after exposure to a tongue graft. Isogenic strains of Brown Norway male rats, weighing 200 to 250 g, were used. An isograft of the sensory vagal nodose ganglion, which consisted of the ganglion and 4 to 6 mm of attached peripheral nerve trunk, was transplanted into the anterior chamber (i.e., the fluid-filled space between the cornea and iris) of the eye of the host rat. One year later an isograft of tongue tissue (taken from a rat different from the one that donated the ganglion) that contained the taste bud-bearing vallate papilla was added to the eye of 10 rats which had a graft of ganglion. The vallate papilla in the graft contained its normal component of taste buds and nerve fibers (i.e., it was not denervated prior to grafting). The tongue graft was adjusted in the eye so that the connective tissue surface of it rested on top of the end of the peripheral nerve trunk of the ganglion. As a control procedure, a 390 0014-4886/80/050390-05$02.00/O
GANGLIA
TRANSPLANTS
391
tongue graft was transplanted alone into the other eye of six of the rats which had combined ganglion-tongue grafts. The techniques of obtaining, trimming, and inserting the grafts were the same as previously reported (11). All grafts were removed 35 days after addition of tongue tissue to the eyes. The ganglia and tongue grafts were separated and each graft was placed between different slabs of skeletal muscle in preparation for freezing in liquid nitrogen. Eight-micrometer-thick frozen cross sections of the grafts were prepared and some stained by periodic acidSchiff hematoxylin (PAS-hematoxylin), adenosine triphosphatase
FIG. I. Normal (A) and transplanted (B) nodose ganglia. Many neurons (one indicated by long arrow) and regions of myelinated nerve fibers (some indicated by short arrow) are present in a normal nodose ganglion (A) whereas reduced numbers of neurons and myelinated nerve fibers are present in a transplanted (B) ganglion. The dark tissue labeled Ir in B is pigmented host iris to which the transplanted ganglion attaches while residing in the eye. PAS-Hematoxylin stain, x 1 IO.
392
ANDREW
A. ZALEWSKI
(ATPase) (7), or cholinesterase (2) procedures. Normal nodose ganglia and vallate papillae were similarly examined. The presence, number, and distribution of neurons and myelinated nerve fibers in a normal nodose ganglion are shown in Fig. 1A. At the time the tongue grafts were added to the eyes, all ganglia were visible and although they were somewhat smaller than normal there was no progressive reduction in size throughout their period of transplantation. Histological examination of the transplanted ganglia revealed that each contained neurons but these were present in reduced numbers (Fig. 1B) compared to a normal ganglion (Fig. 1A). Each transplanted ganglion, however, seemed to have similar numbers of surviving neurons. Many of the surviving neurons exhibited reduced Nissl substance and a displaced nucleus. Myelin staining was demonstrable around nerve fibers in the ganglia but no cellular inflammatory reaction was noted. It is noteworthy that l-year grafts of ganglia had similar numbers of surviving neurons compared to neurons in nodose ganglia grafts that were studied at shorter time periods of previous experiments (8-10). All tongue grafts that were combined with grafts of ganglia had regenerated taste buds (Figs. 2B-D). For comparison purposes, a normal vallate is illustrated in Fig. 2A where it is seen that taste buds are present only in the epithelium of the trench walls of the papilla. In contrast, the regenerated taste buds in tongue grafts were randomly situated in the trench walls (Fig. 2B) and in four grafts some buds were found even in the epithelium on the top surface of the papilla (Fig. 2C). The number of regenerated buds per papilla ranged from 14 to 42. A cholinesterase reaction, indicative of the presence of nerve fibers (9), was always found beneath the epithelium which contained the regenerated taste buds (Fig. 2D). No taste buds were found in any of the six tongue grafts that were transplanted alone (i.e., without ganglion) to eyes (Fig. 2E). The present results demonstrate that sensory neurons can survive longterm transplantation in isolation from their normal tissue. This result contrasts with sympathetic neurons which seem to decrease in number following long transplant periods (4). More importantly, the present study shows that neurons can, after chronic isolation, reinitiate the regeneration of their nerve fibers if they are reexposed to their appropriate tissue. This observation may have implications for reinitiating the regeneration of the injured central nervous system because apparently some nerve fiber growth-stimulating factor(s) is present in grafted tissue. Indeed, Seiger and Olson (5) and Bjorklund and Stenevi (1) have already shown that central nervous system neurons can respond by regeneration if exposed to appropriate tissue grafts. Seiger and Olson (6) also have data which indicate that the growth-promoting factor(s) in grafted tissue is related to
GANGLIA
TRANSPLANTS
393
FIG. 2. Normal vallate papilla (A) and papillae combined with ganglia (B-D) or transplanted alone (E) to eyes. Taste buds are present in the epithelium of the trench walls (arrows) and not in the epithelium on the top surface (indicated by lettering Tp) of a normal papilla (A). Randomly regenerated taste buds are present in the epithelium of the trench walls (arrow of B) and top surface (region bounded by arrows in C) of papillae that were combined with ganglia. A cholinesterase reaction in nerve tissue (arrow of D) is seen beneath the epithelium which supports a regenerated taste bud. No taste buds are present in a papilla transplanted alone to the eye (E). A-C, E. ATPase stain, x70: D. cholinesterase hematoxylin stain, x300.
innervation because grafts of chronically denervated tissue are a poor inducer of axonal outgrowth. It is possible, therefore, that the repair of central nervous system tissue might be accomplished by using a nerve tissue graft (3) and growth factors obtained from grafted tissue. In this scheme, a nerve graft would provide a bridge through which growth factor-stimulated axons might regenerate. REFERENCES I, BJORKLUND. A., AND U. STENEVI. 1979. Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system. Phxsiol. Rev. 59: 62- 100. 2. GOMORI. G. 1952. Microscopic Histochrrnistr~: Principlrs and Practice. Univ. of Chicago Press, Chicago.
394
ANDREW
A. ZALEWSKI
3. KAO, C. C., L. W. CHANG, AND J. M. B. BLOODWORTH, JR. 1977. The mechanism of spinal cord cavitation following spinal cord transection. Part 3. Delayed grafting with and without spinal retransection. J. Neurosurg. 46: 757-766. 4. OLSON, L., AND T. MALMFORS. 1970. Growth characteristics ofadrenergic nerves in the adult rat. Acts Physiol. Stand. Suppl. 348: l-112. 5. SEIGER. A., AND L. OLSON. 1977a. Growth of locus coeruleus neurons in oculo independent of simultaneously present adrenergic and cholinergic nerves in the iris. Med. Biol.
55: 209-223.
6. SEIGER, A., AND OLSON, L. 1977b. Reinitiation of directed nerve fiber growth in central monoamine neurons after intraocular maturation. Exp. Brain Res. 29: 15-44. 7. WACHSTEIN, M., AND E. MEISEL. 1957. Histochemistry of hepatic phosphatase at a physiological pH. Am. J. Clin. Pathol. 27: 13-23. 8. ZALEWSKI, A. A., AND W. K. SILVERS. 1973. Trophic function of neurons in homografts of ganglia in immunologically tolerant rats. &I. Neural. 41: 777-781. 9. ZALEWSKI, A. A. 1976. The neural induction of taste buds in the salivary ducts of the lingual gland of Von Ebner. Exp. Neural. 52: 565-580. 10. ZALEWSKI, A. A., AND W. K. SILVERS. 1977. The long-term fate of neurons in allografts of ganglia in Ag-B-compatible normal and immunologically tolerant rats. J. Neurobiol.
8: 207-215.
11. ZALEWSKI, A. A. 1979. The distribution of alkaline phosphatase activity in normal and cross-species regenerated rat and mouse taste buds. Anat. Rec. 194: 283-292.