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Viability of mature superior cervical ganglia transplants in peripheral nerve of adult rat
Brain Research, 348 (1985) 168-174 Elsevier
168 B R E 21180
Viability of mature superior cervical ganglia transplants in peripheral nerve of adult rat DENNIS W. H O O V L E R l, Y I P E N G TANG 2 and J E R A L D J. BERNSTEIN 1
1Departments of Physiology and Neurosurgery, George Washington University School of Medicine, Washington, D.C. 20037 and Laboratory of Central Nervous System Injury and Regeneration, Veterans Administration Medical Center, Washington, D. C. 20422 (U.S.A.) and 2Visiting Scholar, Central Laboratory, Beijing College of Traditional Chinese Medicine, Beijing (People's Republic of China) (Accepted June 25th, 1985)
Key words: transplantation - - superior cervical ganglion - - peripheral nerve - - fluorescence microscopy
Allografts of mature rat superior cervical ganglia (SCG) survived for up to 120 days following transplantation to regenerating and degenerating peripheral nerves of adult rats. Transplants contained the constituents of normal superior cervical ganglia and there was evidence of outgrowth of catecholamine containing fibers from the transplants. However, fibers did not attain the length necessary to enter peripheral muscles and no muscle response could be elicited by electrical stimulation of the implanted nerve where the transplanted mature SCG was the only source of nerve fibers.
Experiments in the transplantation of nervous tissue from m a t u r e or fetal animals into the central or peripheral nervous systems of adult m a m m a l s have recently met with much success. The basis for such experiments is the possible r e p l a c e m e n t , by the transplanted tissue, of cells and tissue of the nervous system lost to injury or disease. Fetal brain tissue t r a n s p l a n t e d into various areas of adult brain has been shown to survive and integrate with the host central nervous system (CNS) 6, alleviate symptoms associated with an animal m o d e l of Parkinson's diseaseS,14 and correct a b n o r m a l behavior induced by lesion of the adult CNS 9. Transplants of fetal cortex and spinal cord also survive, differentiate and integrate with surrounding host tissue in the adult rat spinal cord, although not to as great a degree as in the brain2,7.13. Recent studies have dealt with the transplantation of fetal CNS tissue into peripheral nerves as a method of augmenting function of d a m a g e d spinal cordl3,4, lO,15. Spinal cord injury causes the loss of functional conduction and trophic influence to peripherally innervated tissues below the site of injury
leading to paralysis and a t r o p h y of muscle tissue. The successful transplantation of viable neurons into the spinal cord or peripheral nerve, and their subsequent afferent and efferent connections to viable host tissue, has the potential of maintaining the integrity of, or possibly restoring function to, functionally denervated tissues. This p r o c e d u r e involves the identification of tissues capable of surviving transplantation, being innervated by host fibers and elaborating axons that have the ability to innervate target tissue. The purpose of the present study was to examine the viability and m o r p h o l o g y of m a t u r e superior cervical ganglia ( S C G ) after transplantation into regenerating or degenerating p e r i p h e r a l nerves of adult rat hosts. M a t u r e S C G was chosen as the transplant tissue because it has been shown to be viable in other transplant studies12,16-19, it is a relatively simple neural tissue that has the potential of being innervated by regenerating fibers 12 and its fiber outgrowth can be evaluated by fluorescence microscopy of catecholaminesS, due to the n o r a d r e n e r g i c nature of its neurons. Sixty adult male S p r a g u e - D a w l e y rats (250-300
Correspondence: D.W. Hoovler, Laboratory of CNS Injury and Regeneration (151Q), 50 Irving Street, N.W., Veterans Administration Medical Center, Washington, D.C. 20422, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
169 g) were anesthetized (Chloropent, 180 mg/kg b.w.) and the nerve to the biceps femoris muscle, exposed. In both the degenerating and regenerating nerve models, the nerve to the biceps femoris was crushed a p p r o x i m a t e l y 1.5 cm from its origin and a small longitudinal slit was m a d e in the e p i n e u r i u m at the crush site. A piece of mature, d e s h e a t h e d S C G was then inserted through the slit with jeweler's forceps, as previously described 4. In the degenerating m o d e l , the sciatic nerve was transected at the level of the sciatic notch and both proximal and distal stumps sutured closed. The proximal stump of the transected sciatic nerve was sutured into a cavity m a d e in the gluteus medius muscle to guard against any possibility of regeneration. Control animals were p r e p a r e d as described above with no SCG transplants. A t 14, 30, 60, 90, or 120 days postoperative ( D P O ) , the nerve to the biceps femoris in host rats (12 at each D P O : 4 experimental and 2 control of both regenerating and degenerating models) was stimulated (5 V, 0.3 ms, 1 Hz) with a bipolar electrode above, below and at the transplant site and note m a d e of any muscle movement. The portion of the nerve to the biceps femoris containing the transplant and the p o r t i o n of the biceps femoris muscle directly innervated by the nerve were processed for catecholamine specific fluorescence microscopy8 and light microscopy. Transplanted m a t u r e S C G neurons were present in both degenerating and regenerating nerves in 68%
Fig. 1. Fluorescent micrograph of normal mature SCG consisting of large, fluorescent sympathetic neurons containing an area of non-fluorescence occupied by the nucleus (arrows), a group of small intensely fluorescent (SIF) cells (arrowhead) and a network of varicose, fluorescent fibers. Glyoxylic acid reaction, x200.
Fig. 2 Fluorescent micrograph of normal nerve to the biceps femoris muscle. Note lack of fluorescent fibers in the nerve and compare with intense fluorescence of the adrenergic innervation of a blood vessel (arrow). Glyoxylic acid reaction, x200.
of the host animals. In general, transplant viability was better at earlier p o s t o p e r a t i v e periods. There was little difference in viability of t r a n s p l a n t e d tissue between regenerating (61%) and degenerating (75%) peripheral nerve models. Control SCG from normal adult rats processed for fluorescence microscopy consisted mainly of large postganglionic sympathetic neurons with fluorescent
Figs. 3-8. Fluorescent micrographs of transplants of mature SCG into regenerating and degenerating nerves to the biceps femoris muscle at various days postoperative. All glyoxylic acid reactions. Fig. 3. 14 DPO regenerating nerve. Transplant has general ovoid shape and contains fluorescent ganglionic neurons (arrows) and fluorescent fibers forming a network around the cells with some appearing to project from the implant (between arrowheads), x 105.
170
Fig. 4. 90 DPO degenerating nerve. Ovoid shaped transplant containing ganglionic neurons (arrows) and a large bundle of fluorescent fibers (between arrowheads), x 105.
Fig. 6.60 DPO degenerating nerve. Transplant containing ganglionic neurons, SIF cells (arrow) and a large blood vessel (between arrowheads), x200.
cytoplasm surrounding an area of non-fluorescence occupied by the nucleus, scattered groups of small intensely fluorescent (SIF) cells and a n e t w o r k of varicose fibers, often in the form of small bundles (Fig. 1). The m a j o r i t y of fluorescent fibers o b s e r v e d in normal peripheral nerves were identified as the adrenergic innervation of blood vessels, identified by their bright fluorescence and tubular profiles (Fig. 2). Transplanted m a t u r e S C G was o b s e r v e d in the nerve to the biceps femoris muscle at most p o s t o p e r ative time periods. The transplants, usually ovoid or spherical in shape, contained distinct groups of fluorescent sympathetic neurons s u r r o u n d e d by a matrix of fluorescent varicose fibers (Fig. 3, 4). The gangli-
onic neurons were similar in size and shape to those seen in control S C G and the intensity and distribution of fluorescence also a p p e a r e d normal. The fibers accompanying the neurons were brightly fluorescent, contained fluorescent varicosities and f o r m e d an intermingling n e t w o r k (Fig. 5). Occasionally, however, fluorescent fibers similar to those surrounding the ganglionic neurons and atypical of those seen in normal nerve were observed at greater distances from the neurons and running parallel to the p e r i p h e r a l nerve (Figs. 3, 4). B l o o d vessels and groups of SIF cells were also observed in the transplants (Figs. 6, 7). In transplants at 120 D P O , some ganglionic neurons showed a diffuse fluorescent a p p e a r a n c e and a
Fig. 5. 14 DPO regenerating nerve. Higher magnification of Fig. 3 showing network of varicose fluorescent fibers surrounding the ganglionic neurons, x200.
Fig. 7.90 DPO degenerating nerve. Transplant containing extensive network of fluorescent fibers and a group of SIF cells (arrow). x200.
171
Fig. 8. 120 DPO degenerating nerve. Transplanted neurons after 4 months had a light, diffuse fluorescence, possibly a sign of degeneration (arrowheads). Note small pyknotic cells (arrows), x200.
number of small pyknotic cells containing fluorescent granules were present (Fig. 8). No fluorescent fibers, except for those associated with blood vessels, were observed in muscle tissue innervated by regenerating or degenerating control or implanted nerves. Control SCG from normal adult rats processed for light microscopy showed the ganglion to consist of large postganglionic sympathetic neurons, satellite cells and blood vessels. Normal ganglionic neurons exhibited a large nuclear to cytoplasmic ratio (large nucleus, little cytoplasm) with light homogeneous nuclei, some containing 2 nucleoli, surrounded by a
Fig. 9. Light micrograph of normal mature SCG consisting of large sympathetic neurons, some containing 2 nucleoli (arrows), small satellite cells and blood vessels (v). Hematoxylin and eosin stain, x200.
Figs. 10-14. Light micrographs of transplants of mature SCG into regenerating and degenerating nerve to the biceps femoris muscle at various days postoperative. All hematoxylin and eosin stain. Blood vessel (v). Fig. 10. 14 days postoperative (DPO) regenerating nerve. Nuclei of transplanted SCG neurons appear small and eccentric suggesting a chromatolytic response. Blood vessel (v). x200.
slightly darker rim of granular cytoplasm (Fig. 9). In both the regenerating and degenerating nerve to the biceps femoris muscle, transplants of SCG were spherical or ovoid in shape and contained support cells, blood vessels and varying numbers (5-50) of ganglionic neurons, (Figs. 10-14). At earlier postoperative periods (14-30 D P O ) , changes in nuclear density and position and smaller nuclear to cytoplasmic ratios were present. At later periods (60-120
Fig. 11.30 DPO degenerating nerve. Transplanted neurons appear similar to those of normal ganglion, x200.
172
Fig. 12. 60 DPO degenerating nerve. Transplanted neurons contain small, dark nuclei and dark cytoplasm indicating cellular necrosis. ×200.
Fig. 14. 120 DPO regenerating nerve. Transplanted neurons appear very similar to those in normal ganglion after 4 months, although one may contain 2 nuclei (arrow). ×200.
DPO), various transplants showed signs of cellular necrosis including darkened and shrunken nuclei and cytoplasm. However, some transplanted ganglion cells retained a normal morphology up to 120 D P O (Fig. 14). In the degenerating model animals, where the sciatic nerve had been transected, the biceps femoris muscle fibers were small and separated by adipose tissue in both transplanted and control nerves. In the regenerating model animals, both transplanted and control, muscle tissue appeared normal. Stimulation of control nerves and nerves containing the transplants did not result in the contraction of the biceps femoris muscle in any animals in which the sciatic nerve was transected (the degener-
ating model). Stimulation of control and implanted nerves in regenerating model animals resulted in contraction of the biceps femoris muscle in all cases. These results generally agree with other studies on the viability of mature SCG when transplanted to various other sites. Mature SCG transplanted to the anterior chamber of the eye survives at least 3 weeks and elaborates fibers into the previously denervated host's iris TM. Mature SCG survives and is innervated by substance P immunoreactive fibers 2 weeks after transplantation into the spinal cord 12. Longer term studies show that, after a period of degeneration, some transplanted mature SCG neurons survive up to 6 months on the pial covering in the choroidal fissure 19 and up to 2.5 years in the fourth ventricle iv. Long term viability of transplanted mature SCG appears to depend mainly on vascularization of the transplant. Mature SCG transplanted into brain parenchyma exhibit low viability 19 whereas SCG transplanted to brain surfaces rich in blood vessels thrives 16,I9. Secondly, it has been suggested that long term viability of transplanted mature SCG is dependent upon the presence of target tissue connectivity 17. In the present study, transplants appeared to be well vascularized but there was no morphological or electrophysiological evidence that the biceps femoris muscle had been reinnervated by fibers from the transplants. Evidence of fiber outgrowth was observed in the present study but not to any great extent. This could be due to a lack of sufficient time for extensive elongation, presence or regrowth of a por-
Fig. 13. 90 DPO regenerating nerve. Small, dark nuclei and dark cytoplasm again indicate cellular necrosis, x 200.
173 tion of the connective tissue capsule, lack of a suitable e n v i r o n m e n t for elongation, too great a distance to traverse before establishment of connectivity or possibly that individual fibers did elongate but their
have terminals identified as growth cones as late as 6 months after transplantation 17. These fibers form a mesh-work throughout the transplant and account for the abundance of fluorescent fibers surrounding
presence was too subtle to detect with the method employed.
the implanted postganglionic n e u r o n s seen here and in the study of Stenevi, et al. 19.
Transplantation of mature SCG necessitates the loss of postganglionic efferent target site connections as well as the loss of afferent preganglionic cholinergic input to the postganglionic neurons. The former is reported to lead to reversible detachment of pregan-
Fluorescent microscopic results indicated that transplanted mature SCG contained and continued to produce noradrenaline, which is responsible for
glionic synapses from ganglionic n e u r o n s l l , while the latter induces a period of degeneration and subse-
transplants in the brain has been shown by fluorescence microscopy t9 and by using various electron mi-
quent chromatolysis of the postganglionic neurons17. Degenerating transplanted mature SCG n e u r o n s were prevalent in the present study and in other studies a6,19 and are the result of lost connectivity. A sur-
croscopic markers for catecholamines 17. Fluorescence microscopy also showed the presence of SIF cells in the transplants, which have been shown to
vival rate of 1 - 2 % was reported in the fluorescence study of Stenevi, et a1.19. They saw large areas of the transplant devoid of cells and 'small pyknotic cells filled with autofluorescent granular material' as in the present study. However, some ganglionic neurons do survive and sprout n u m e r o u s processes which
1 Bernstein, J.J., Viability, growth and maturation of fetal cortex and spinal cord in the sciatic nerve of adult rat, J. Neurosci. Res., 10 (1983) 343-350. 2 Bernstein, J.J., Patel, U., Kelemen, M., Jefferson, M. and Turtil, S., Ultrastructure of fetal spinal cord and cortex implants into adult rat spinal cord, J. Neurosci. Res., 11 (1984) 359-372. 3 Bernstein, J.J. and Tang Y., Structure and function of fetal cortex implanted into the peripheral nerve of adult rat, Brain Research, 324 (1984) 243-251. 4 Bernstein, J.J., Tang, Y. and Hoovler, D.W., Implantation of fetal CNS into peripheral nerve. In A Bj6rklund and U. Stenevi (Eds.), Neural Grafting in the Mammalian CNS, Elsevier, Amsterdam, 1985, pp. 329-334. 5 Bj6rklund, A. Stenevi, U., Dunnen, S.B. and Iversen, S.D., Functional reactivation of the deafferented neostriaturn by nigral transplants, Nature (London), 289 (1981) 497-499. 6 Bj6rklund, A., Stenevi, U. and Svendgaard, N., Growth of transplanted monoaminergic neurons into the adult hippocampus along the perforant path, Nature (London), 262 (1976) 787-790. 7 Das, G.D., Neural transplantation in the spinal cord of the adult mammal. In C. Kao, R. Bunge and P. Reir (Eds.), Spinal Cord Reconstruction, Raven Press, New York, 1983, pp. 367-396. 8 de la Torre, J.C. and Surgeon, J.W., A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylicacid technique: the SPG method, Histochemistry, 49 (1976) 81-93.
the catecholamine specific fluorescence. The production and presence of n o r a d r e n a l i n e in mature SCG
survive transplantation in a previous fluorescence study 19 but which were not observed in an electron microscopic study16. Research supported by the A m e r i c a n Paralysis Association and the Veterans Administration. The authors thank Dr. J. C o n n o r for contributions to the manuscript.
9 Dunnen, S.B., Low, W.C., Iversen, S.D., Stenevi, U. and Bj6rklund, A., Septal transplants restore maze learning in rats with fornix-fimbria lesions, Brain Research, 251 (1982) 335-348. 10 Hoovler, D.W. and Bernstein, J.J., Transplantation of fetal rat cortex into regenerating nerve to the biceps femoris of adult rat, Exp. Neurol., 89 (1985) 337-347. 11 Matthews, M.R. and Nelson, V.H., Detachment of structurally intact nerve endings from chromatolytic neurones of rat superior cervical ganglion during the depression of synaptic transmission induced by post-ganglionic axotomy, J. Physiol., (London), 245 (1975) 91-135. 12 Mitta, M., Nakamura, M, Kohno, J., Ono, K., Shiosaka, S., Sakanaka, M., Yamasaki, H., Nakamura, S., Tohyama, M., Powell, J.F. and Smith, A.D., Growth of central substance P-containing neurons into superior cervical ganglia transplanted in the spinal cord of adult rats, Brain Research, 324 (1984) 134-137. 13 Patel, U. and Bernstein, J.J., Growth, differentiation and viability of fetal rat cortical and spinal cord implants into adult rat spinal cord, J. Neurosci. Res., 9 (1983) 303-310. 14 Perlow, M.J., Freed, W.J., Hoffer, B.J., Seiger, A., O1son, L. and Wyatt, R.J., Brain grafts reduce motor abnormalities produced by destruction of the nigrostriatal dopamine system, Science, 204 (1979) 643-647. 15 Richardson, P. and Issa, V., Transplantation of embryonic spinal and cerebral tissue to sciatic nerves of adult rats, Brain Research, 298 (1984) 146-148. 16 Rosenstein, J.M. and Brightman, M.W., Regeneration and myelination in autonomic ganglia transplanted to intact
174 brain surfaces, J. Neurocytol., 8 (1979) 359-379. 17 Rosenstein, J.M. and Brightman, M.W., Some consequences of grafting autonomic ganglia to brain surfaces. In J.R. Sladek, Jr. and D.M. Gash (Eds.), Neural Transplants, Plenum, 1984, pp. 423-443. 18 Seiger, A. and Olson, L., Growth of locus coeruleus neurons in oculo independent of simultaneously present adren-
ergic and cholinergic nerves in the iris, Med. Biol., 55 (1977) 209-223. 19 Stenevi, U., Bj6rklund, A. and Svendgaard, N.A., Transplantation of central and peripheral monoamine neurons to the adult rat brain: techniques and conditions for survival, Brain Research, 114 (1976) 1-20.
168 B R E 21180
Viability of mature superior cervical ganglia transplants in peripheral nerve of adult rat DENNIS W. H O O V L E R l, Y I P E N G TANG 2 and J E R A L D J. BERNSTEIN 1
1Departments of Physiology and Neurosurgery, George Washington University School of Medicine, Washington, D.C. 20037 and Laboratory of Central Nervous System Injury and Regeneration, Veterans Administration Medical Center, Washington, D. C. 20422 (U.S.A.) and 2Visiting Scholar, Central Laboratory, Beijing College of Traditional Chinese Medicine, Beijing (People's Republic of China) (Accepted June 25th, 1985)
Key words: transplantation - - superior cervical ganglion - - peripheral nerve - - fluorescence microscopy
Allografts of mature rat superior cervical ganglia (SCG) survived for up to 120 days following transplantation to regenerating and degenerating peripheral nerves of adult rats. Transplants contained the constituents of normal superior cervical ganglia and there was evidence of outgrowth of catecholamine containing fibers from the transplants. However, fibers did not attain the length necessary to enter peripheral muscles and no muscle response could be elicited by electrical stimulation of the implanted nerve where the transplanted mature SCG was the only source of nerve fibers.
Experiments in the transplantation of nervous tissue from m a t u r e or fetal animals into the central or peripheral nervous systems of adult m a m m a l s have recently met with much success. The basis for such experiments is the possible r e p l a c e m e n t , by the transplanted tissue, of cells and tissue of the nervous system lost to injury or disease. Fetal brain tissue t r a n s p l a n t e d into various areas of adult brain has been shown to survive and integrate with the host central nervous system (CNS) 6, alleviate symptoms associated with an animal m o d e l of Parkinson's diseaseS,14 and correct a b n o r m a l behavior induced by lesion of the adult CNS 9. Transplants of fetal cortex and spinal cord also survive, differentiate and integrate with surrounding host tissue in the adult rat spinal cord, although not to as great a degree as in the brain2,7.13. Recent studies have dealt with the transplantation of fetal CNS tissue into peripheral nerves as a method of augmenting function of d a m a g e d spinal cordl3,4, lO,15. Spinal cord injury causes the loss of functional conduction and trophic influence to peripherally innervated tissues below the site of injury
leading to paralysis and a t r o p h y of muscle tissue. The successful transplantation of viable neurons into the spinal cord or peripheral nerve, and their subsequent afferent and efferent connections to viable host tissue, has the potential of maintaining the integrity of, or possibly restoring function to, functionally denervated tissues. This p r o c e d u r e involves the identification of tissues capable of surviving transplantation, being innervated by host fibers and elaborating axons that have the ability to innervate target tissue. The purpose of the present study was to examine the viability and m o r p h o l o g y of m a t u r e superior cervical ganglia ( S C G ) after transplantation into regenerating or degenerating p e r i p h e r a l nerves of adult rat hosts. M a t u r e S C G was chosen as the transplant tissue because it has been shown to be viable in other transplant studies12,16-19, it is a relatively simple neural tissue that has the potential of being innervated by regenerating fibers 12 and its fiber outgrowth can be evaluated by fluorescence microscopy of catecholaminesS, due to the n o r a d r e n e r g i c nature of its neurons. Sixty adult male S p r a g u e - D a w l e y rats (250-300
Correspondence: D.W. Hoovler, Laboratory of CNS Injury and Regeneration (151Q), 50 Irving Street, N.W., Veterans Administration Medical Center, Washington, D.C. 20422, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
169 g) were anesthetized (Chloropent, 180 mg/kg b.w.) and the nerve to the biceps femoris muscle, exposed. In both the degenerating and regenerating nerve models, the nerve to the biceps femoris was crushed a p p r o x i m a t e l y 1.5 cm from its origin and a small longitudinal slit was m a d e in the e p i n e u r i u m at the crush site. A piece of mature, d e s h e a t h e d S C G was then inserted through the slit with jeweler's forceps, as previously described 4. In the degenerating m o d e l , the sciatic nerve was transected at the level of the sciatic notch and both proximal and distal stumps sutured closed. The proximal stump of the transected sciatic nerve was sutured into a cavity m a d e in the gluteus medius muscle to guard against any possibility of regeneration. Control animals were p r e p a r e d as described above with no SCG transplants. A t 14, 30, 60, 90, or 120 days postoperative ( D P O ) , the nerve to the biceps femoris in host rats (12 at each D P O : 4 experimental and 2 control of both regenerating and degenerating models) was stimulated (5 V, 0.3 ms, 1 Hz) with a bipolar electrode above, below and at the transplant site and note m a d e of any muscle movement. The portion of the nerve to the biceps femoris containing the transplant and the p o r t i o n of the biceps femoris muscle directly innervated by the nerve were processed for catecholamine specific fluorescence microscopy8 and light microscopy. Transplanted m a t u r e S C G neurons were present in both degenerating and regenerating nerves in 68%
Fig. 1. Fluorescent micrograph of normal mature SCG consisting of large, fluorescent sympathetic neurons containing an area of non-fluorescence occupied by the nucleus (arrows), a group of small intensely fluorescent (SIF) cells (arrowhead) and a network of varicose, fluorescent fibers. Glyoxylic acid reaction, x200.
Fig. 2 Fluorescent micrograph of normal nerve to the biceps femoris muscle. Note lack of fluorescent fibers in the nerve and compare with intense fluorescence of the adrenergic innervation of a blood vessel (arrow). Glyoxylic acid reaction, x200.
of the host animals. In general, transplant viability was better at earlier p o s t o p e r a t i v e periods. There was little difference in viability of t r a n s p l a n t e d tissue between regenerating (61%) and degenerating (75%) peripheral nerve models. Control SCG from normal adult rats processed for fluorescence microscopy consisted mainly of large postganglionic sympathetic neurons with fluorescent
Figs. 3-8. Fluorescent micrographs of transplants of mature SCG into regenerating and degenerating nerves to the biceps femoris muscle at various days postoperative. All glyoxylic acid reactions. Fig. 3. 14 DPO regenerating nerve. Transplant has general ovoid shape and contains fluorescent ganglionic neurons (arrows) and fluorescent fibers forming a network around the cells with some appearing to project from the implant (between arrowheads), x 105.
170
Fig. 4. 90 DPO degenerating nerve. Ovoid shaped transplant containing ganglionic neurons (arrows) and a large bundle of fluorescent fibers (between arrowheads), x 105.
Fig. 6.60 DPO degenerating nerve. Transplant containing ganglionic neurons, SIF cells (arrow) and a large blood vessel (between arrowheads), x200.
cytoplasm surrounding an area of non-fluorescence occupied by the nucleus, scattered groups of small intensely fluorescent (SIF) cells and a n e t w o r k of varicose fibers, often in the form of small bundles (Fig. 1). The m a j o r i t y of fluorescent fibers o b s e r v e d in normal peripheral nerves were identified as the adrenergic innervation of blood vessels, identified by their bright fluorescence and tubular profiles (Fig. 2). Transplanted m a t u r e S C G was o b s e r v e d in the nerve to the biceps femoris muscle at most p o s t o p e r ative time periods. The transplants, usually ovoid or spherical in shape, contained distinct groups of fluorescent sympathetic neurons s u r r o u n d e d by a matrix of fluorescent varicose fibers (Fig. 3, 4). The gangli-
onic neurons were similar in size and shape to those seen in control S C G and the intensity and distribution of fluorescence also a p p e a r e d normal. The fibers accompanying the neurons were brightly fluorescent, contained fluorescent varicosities and f o r m e d an intermingling n e t w o r k (Fig. 5). Occasionally, however, fluorescent fibers similar to those surrounding the ganglionic neurons and atypical of those seen in normal nerve were observed at greater distances from the neurons and running parallel to the p e r i p h e r a l nerve (Figs. 3, 4). B l o o d vessels and groups of SIF cells were also observed in the transplants (Figs. 6, 7). In transplants at 120 D P O , some ganglionic neurons showed a diffuse fluorescent a p p e a r a n c e and a
Fig. 5. 14 DPO regenerating nerve. Higher magnification of Fig. 3 showing network of varicose fluorescent fibers surrounding the ganglionic neurons, x200.
Fig. 7.90 DPO degenerating nerve. Transplant containing extensive network of fluorescent fibers and a group of SIF cells (arrow). x200.
171
Fig. 8. 120 DPO degenerating nerve. Transplanted neurons after 4 months had a light, diffuse fluorescence, possibly a sign of degeneration (arrowheads). Note small pyknotic cells (arrows), x200.
number of small pyknotic cells containing fluorescent granules were present (Fig. 8). No fluorescent fibers, except for those associated with blood vessels, were observed in muscle tissue innervated by regenerating or degenerating control or implanted nerves. Control SCG from normal adult rats processed for light microscopy showed the ganglion to consist of large postganglionic sympathetic neurons, satellite cells and blood vessels. Normal ganglionic neurons exhibited a large nuclear to cytoplasmic ratio (large nucleus, little cytoplasm) with light homogeneous nuclei, some containing 2 nucleoli, surrounded by a
Fig. 9. Light micrograph of normal mature SCG consisting of large sympathetic neurons, some containing 2 nucleoli (arrows), small satellite cells and blood vessels (v). Hematoxylin and eosin stain, x200.
Figs. 10-14. Light micrographs of transplants of mature SCG into regenerating and degenerating nerve to the biceps femoris muscle at various days postoperative. All hematoxylin and eosin stain. Blood vessel (v). Fig. 10. 14 days postoperative (DPO) regenerating nerve. Nuclei of transplanted SCG neurons appear small and eccentric suggesting a chromatolytic response. Blood vessel (v). x200.
slightly darker rim of granular cytoplasm (Fig. 9). In both the regenerating and degenerating nerve to the biceps femoris muscle, transplants of SCG were spherical or ovoid in shape and contained support cells, blood vessels and varying numbers (5-50) of ganglionic neurons, (Figs. 10-14). At earlier postoperative periods (14-30 D P O ) , changes in nuclear density and position and smaller nuclear to cytoplasmic ratios were present. At later periods (60-120
Fig. 11.30 DPO degenerating nerve. Transplanted neurons appear similar to those of normal ganglion, x200.
172
Fig. 12. 60 DPO degenerating nerve. Transplanted neurons contain small, dark nuclei and dark cytoplasm indicating cellular necrosis. ×200.
Fig. 14. 120 DPO regenerating nerve. Transplanted neurons appear very similar to those in normal ganglion after 4 months, although one may contain 2 nuclei (arrow). ×200.
DPO), various transplants showed signs of cellular necrosis including darkened and shrunken nuclei and cytoplasm. However, some transplanted ganglion cells retained a normal morphology up to 120 D P O (Fig. 14). In the degenerating model animals, where the sciatic nerve had been transected, the biceps femoris muscle fibers were small and separated by adipose tissue in both transplanted and control nerves. In the regenerating model animals, both transplanted and control, muscle tissue appeared normal. Stimulation of control nerves and nerves containing the transplants did not result in the contraction of the biceps femoris muscle in any animals in which the sciatic nerve was transected (the degener-
ating model). Stimulation of control and implanted nerves in regenerating model animals resulted in contraction of the biceps femoris muscle in all cases. These results generally agree with other studies on the viability of mature SCG when transplanted to various other sites. Mature SCG transplanted to the anterior chamber of the eye survives at least 3 weeks and elaborates fibers into the previously denervated host's iris TM. Mature SCG survives and is innervated by substance P immunoreactive fibers 2 weeks after transplantation into the spinal cord 12. Longer term studies show that, after a period of degeneration, some transplanted mature SCG neurons survive up to 6 months on the pial covering in the choroidal fissure 19 and up to 2.5 years in the fourth ventricle iv. Long term viability of transplanted mature SCG appears to depend mainly on vascularization of the transplant. Mature SCG transplanted into brain parenchyma exhibit low viability 19 whereas SCG transplanted to brain surfaces rich in blood vessels thrives 16,I9. Secondly, it has been suggested that long term viability of transplanted mature SCG is dependent upon the presence of target tissue connectivity 17. In the present study, transplants appeared to be well vascularized but there was no morphological or electrophysiological evidence that the biceps femoris muscle had been reinnervated by fibers from the transplants. Evidence of fiber outgrowth was observed in the present study but not to any great extent. This could be due to a lack of sufficient time for extensive elongation, presence or regrowth of a por-
Fig. 13. 90 DPO regenerating nerve. Small, dark nuclei and dark cytoplasm again indicate cellular necrosis, x 200.
173 tion of the connective tissue capsule, lack of a suitable e n v i r o n m e n t for elongation, too great a distance to traverse before establishment of connectivity or possibly that individual fibers did elongate but their
have terminals identified as growth cones as late as 6 months after transplantation 17. These fibers form a mesh-work throughout the transplant and account for the abundance of fluorescent fibers surrounding
presence was too subtle to detect with the method employed.
the implanted postganglionic n e u r o n s seen here and in the study of Stenevi, et al. 19.
Transplantation of mature SCG necessitates the loss of postganglionic efferent target site connections as well as the loss of afferent preganglionic cholinergic input to the postganglionic neurons. The former is reported to lead to reversible detachment of pregan-
Fluorescent microscopic results indicated that transplanted mature SCG contained and continued to produce noradrenaline, which is responsible for
glionic synapses from ganglionic n e u r o n s l l , while the latter induces a period of degeneration and subse-
transplants in the brain has been shown by fluorescence microscopy t9 and by using various electron mi-
quent chromatolysis of the postganglionic neurons17. Degenerating transplanted mature SCG n e u r o n s were prevalent in the present study and in other studies a6,19 and are the result of lost connectivity. A sur-
croscopic markers for catecholamines 17. Fluorescence microscopy also showed the presence of SIF cells in the transplants, which have been shown to
vival rate of 1 - 2 % was reported in the fluorescence study of Stenevi, et a1.19. They saw large areas of the transplant devoid of cells and 'small pyknotic cells filled with autofluorescent granular material' as in the present study. However, some ganglionic neurons do survive and sprout n u m e r o u s processes which
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the catecholamine specific fluorescence. The production and presence of n o r a d r e n a l i n e in mature SCG
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