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Localization and changes of substance P in spinal cord of paraplegic cats
Brain Research, 153 (1978) 507-513 ~ Elsevier/North-Holland Biomedical Press
507
L O C A L I Z A T I O N A N D C H A N G E S OF SUBSTANCE P I N S P I N A L C O R D OF P A R A P L E G I C CATS
N. E. NAFTCHI, S. J. A B R A H A M S , H. M. ST. PAUL, E. W. LOWMAN and W. SCHLOSSER
Laboratory of Biochemical Pharmacology, lnstHute of Rehabilitation Medicine, New York University Medical Center, 400 East 34th Street, New York, N. Y. 10016and (W.S.) Hoffmann-La Roche, Research Division, Nutley, N.J. 07110 (U.S.A.)
(Accepted January 18th, 1978)
SUMMARY Female cats were transected at the T12 region of the spinal cord and sacrificed by perfusion through the aorta after 5 days, 2, 5, 8 or 12 weeks. Tissue from above and below the lesion was postfixed, dehydrated and embedded in paraffin. 5 # m sections were incubated for substance P immunoreactivity by the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Spinal cord sections of normal cats showed a small amount of peroxidase-positive staining in the dorsal horn in laminae I, II and III of Rexed. Starting at 5 days after transection, there was a sharp increase in the amount of substance P-staining below the lesion in the form of punctate bodies and long varicosities. Above the lesion in the region of the substantia gelatinosa the number of stained bodies was fewer and the intensity of staining around the dorsal horns was less than that in sections obtained from below the lesion. This pattern was repeated when sections from animals sacrificed at later times were stained for substance P. The results confirm the presence of substance P in the spinal cord nerve tracts. Its accumulation in the dorsal horn below the transection lends credence to its role as a transmitter or modulator of afferent neuronal activity, and suggests its rostral direction by axoplasmic flow.
INTRODUCTION In 1931, Von Euler and Gaddum extracted a factor from equine brain and intestine which they called substance p21. It was found to possess a vasodilatory effect and stimulate smooth muscle. It was not until 1970 when Chang and Leeman 2 isolated a sialogogic peptide from bovine hypothalamus that it was finally purified, characterized as substance P, synthesized, and its regional distribution studied by radio-
508 immunoassay and immunofluorescence techniquesa, z,~8. Leeman and coworkers have shown substance P to be an undecapeptide 2. It has an uneven distribution in the CNS. Its highest concentrations are found in the substantia nigra, hypothalamus, pineal gland and the dorsal gray matter of the spinal cord1,'4, 9. When applied directly to single neurons, such as the motor neuron of the ventral horn of the spinal cord, it provokes a slow, long-lasting excitatory response tS. It has been localized by immunofluorescence in the substantia gelatinosa of the dorsal horn of human spinal cord by Cuello et al. 4, and in the same area of the cat and the rat by H6kfelt ct al.S,% The immunoreactive fibers seem to represent thin unmyelinated fibers classically known to carry thermal and pain stimuli. The latter finding suggests that substance P is the first chemical signal for exteroceptive perception in the human spinal cord '1. In this study. we have localized substance P in the spinal cords of cats by the peroxidase-antiperoxidase (PAP) immunocytochemical technique, and have followed its distribution after the transection of the spinal cord. A preliminary report of our findings has been published 13. MATERIALS AND METHODS
Preparation of animals Female cats weighing between 2.1 and 3.2 kg were selected and anesthetized with 30 mg/kg ketamine, i.m. A dorsal laminectomy was performed and the spinal cord was exposed and transected at T12. Small pieces of sterile gelfoam were placed between the cut ends of the cord in the cats which immediately stopped any bleeding that had occurred. The gelfoam was left in place and the animals were sutured. Animals were not fed or watered for one day following surgery, after which they were allowed to eat or drink ad libitum, and their bladders were expressed 3 times daily.
Tissue preparation At varying time intervals after transection, the animals were anesthetized with Nembutal and sacrificed by perfusion through the ascending aorta with 4 ~ paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 for 10 min in. Sections approximately 2 cm long were removed from above and below the lesion in the spinal cord. These pieces were immediately immersed in picric acid-formaldehyde fixative for about 6 h, after which time they were cut into smaller pieces. The tissues were then rinsed in phosphate-buffered saline from 2 h to overnight, dehydrated through a graded series of ethanol solutions, put through several changes of xylene and paraffin, and finally embedded in paraffin. Sections were cut at a thickness of 4-5 # m and affixed to albumin-coated glass slides. Once prepared, these slides could be incubated for immunoreactivity at any future time.
Antisera Rabbit antiserum to substance P was generously given to us b y Dr. Susan Leeman. Prior to incubation of the tissue, the antiserum was diluted 1:100 with 0.5 M
509 Tris.saline (pH 7.6) and incubated for 2 h at 37 °C with rat liver acetone powder (20 mg liver powder/ml of diluted serum). This was stored overnight at 4 °C and filtered the next morning through a 0.2 #m millipore filter. It was then further diluted with 0.5 M Tris.saline. Final dilutions used in this series of experiments ranged from 1:400 to 1 : 1000.
Immunohistochemical staining The peroxidase-anti-peroxidase immunohistochemical method as first described by Sternberger 2° and modified by Pickel et al. iv was performed on 5/~m tissue sections for localization of substance P in the cat tissue. Non-specific protein binding was reduced by incubating the sections with 3 goat serum in Tris.saline before they were incubated with anti-substance P antiserum for 1 h. Goat anti-rabbit immunoglobulin (Miles Labs) was applied for 30 min after two brief rinses with Tris.saline. Following two more rinses, the PAP reagent (Dako Accurate Chemicals) was applied in 1:25 dilution for 30 min. The slides were then incubated for 15-30 min with 0 . 0 5 ~ 3,3'-diaminobenzidine (Sigma) and 0.01~,i hydrogen peroxide solution in Tris buffer (pH 7.6). A brown precipitate was formed characteristic of polymerized diaminobenzidine. After several washes in distilled water, the slides were dehydrated and mounted with coverslips. Sham-operated animals served as controls. Tissues from these animals were processed in the same manner as above. In order to control for immunohistochemical specificity, normal rabbit serum was routinely substituted for specific rabbit antibody to substance P in all experiments. The sections were examined with a Jena light microscope at magnifications of 25-630 ×. RESULTS Tissue from sham-operated cats (Fig. la and b) displayed a small amount of peroxidase-positive staining which appeared as a band of nerve terminals in the dorsal horn at the junction between the white and grey matters, the substantia gelatinosa which corresponds to laminae II and Ill of Rexed 19. Five days after transection, there was a sharp increase in the amount of substance P below the lesion which had outlined both dorsal horns and bridged the two together. Above the lesion, in the region of the substantia gelatinosa, the number of punctate bodies was fewer, and the intensity of staining around the dorsal horns was less than that in sections obtained from below the lesion. This pattern was repeated when sections from animals sacrificed 1, 2, 5, 8 and 12 weeks after transection were stained for substance P. Specific staining in sections cut from above the lesion (Fig. 2a and 3a), was little compared with the great amount of staining below (Figs. 2b and 3b). In addition to punctate bodies, long fibers and varicosities were present in sections taken from below the lesion (Fig. 2c), and a network of fibers located centrally within each dorsal horn from cats sacrificed 12 weeks after transection was positively stained (Figs. 3b and c). A small amount of immunoreactive product was often seen in the
Fig. I. a: section from TI 1 12 region of the spinal cord of a s h a m - o p e r a t e d cat. Arro~,~ noint to in~m u n o r e a c t i v e s u b s t a n c e P (SP) specific bodies in dorsal horn (area o f s u b s t a n t i a gelatinosa, SGt, 200. (Bar : 100/tin in all figures), b: phase contrast micrograph of the s a m e dorsal h o r n reginri seen in ~. T h e section was i n c u b a t e d for SP immunoreaclivity. Note the presence of a dark ring in the SG regioH, indicating the area of immunoreactivity. 200. c : phase contrast m i c r o g r a p h of the same dorsal h o r n region seen in a. T h e section was incubated with n o r m a l rabbii serum. C o m p a r e d t{~ b. ~,he ~bsence ~f a n y SP-specific staining is evident. 200,
Fig. 2. a: field s h o w i n g dorsal h o r n s abow~ the lesion from a cat transected 5 weeks belk)re perfusion. SG s h o w s slight staining, ;~ 125. b: tissue from the s a m e cat as in a sectioned below the lesion. Note the intense reaction p r o d u c t delineating both dorsal horns. :, 100. c: this is a detail o f the SG seen in b. A r r o w s point to darkly stained S P - i m m u n o r e a c t i v e punctate bodies a n d a n intense band of bead-like varicosities delineating the entire SG, ~ 250. d: section o f the ventral h o r n from below the lesion of a cat transected 5 weeks before sacrifice. Ventral h o r n cells (V) are present. A r r o w s point to possible i m m u n o positive bodies that are scattered t h r o u g h o u t . 250,
511
Fig. 3. a: section from above the lesion of a cat transected 12 weeks before sacrifice. Note sparse staining surrounding the dorsal horn in the SG. x 200. b: tissue from the same cat as in a, sectioned below the lesion. The staining of the band of nerve terminals in the SG is much more intense in this section than that in the section above the lesion. Note the appearance of the nerve plexus (arrow) near the center (Lamina V) of the dorsal horn. x 160. c: a detail of the nerve plexus from b. Immunoreactive substance appears in bead-like, fibrillar varicosities which may be nerve terminals originating from dorsal roots and entering the spinal cord. RBC, red blood cells present in small capillaries. ~. 400. ventral horn in the f o r m o f small, dense p u n c t a t e bodies scattered r a n d o m l y t h r o u g h o u t the horn (Fig. 2d). It was difficult to assess whether the a m o u n t o f staining changed with time either a b o v e or below the lesion in the ventral horn. S u b s t i t u t i o n o f n o r m a l serum for i m m u n e serum as a control showed no staining in the grey m a t t e r o f the spinal cord, with the exception o f red b l o o d cells, which contain e n d o g e n o u s peroxidase (Fig. lc). In some specimens, i m m u n o r e a c t i v e staining did a p p e a r in the white matter, which could be a t t r i b u t e d to some degree o f nonspecific affinity o f the r a b b i t serum for the m e m b r a n e in the region where the myelin had leached out, since the same type of light-brown b a c k g r o u n d staining a p p e a r e d in control sections where n o r m a l r a b b i t serum was substituted for the specific antiserum. There were, however, some small, d a r k punctate bodies present in the myelinated axons o f the white m a t t e r in the ventrolateral and d o r s o l a t e r a l parts o f the spinal cord. This m a y suggest r o s t r a l a x o n a l flow o f substance P within m y e l i n a t e d fibers, possibly belonging to lateral s p i n o t h a l a m i c tracts or to shorter tracts concerned with segmental transmission. DISCUSSION The a b u n d a n c e o f substance P - i m m u n o r e a c t i v e nerve terminals in the substantia gelatinosa suggests t h a t substance P is contained in the p r i m a r y afferent fibers which t e r m i n a t e in the d o r s a l horn. O t s u k a et al. 15 ligated the d o r s a l r o o t s o f the cat and f o u n d that substance P a c c u m u l a t e d on the ganglion side o f the ligature, but its level on the central side decreased. The finding o f substance P in these afferent fibers suggested a role in neurotransmission. W o r k o f Konishi a n d O t s u k a 10 on the isolated
512 frog spinal cord preparation has shown substance P to be about 200 times more active on a molar basis than L-glutamate in depolarizing spinal motor neurons. This depolarizing action persisted after synaptic transmission was blocked by Ca'-'~-deficient Ringer's solution or by tetrodotoxin. They concluded, therefore, that substance P exerted a transsynaptic action on motor neurons, and was probably a candidate for the excitatory transmitter of primary sensory neurons 10,11. Iontophoretic application of substance P to spinal neurons by Henry6, 7 produced a strong but slow and prolonged excitatory action on nearly half the neurons tested in the lumbar spinal cord of the cat. It is noteworthy that all units excited by substance P were also excited by noxious thermal stimulation of the skin. The highest number of units excited by substance P was found in lamina VI and the lowest in lamina IV. In two cases, treatment with substance P led to a response to noxious heat by units which had previously been unresponsive to thermal stimulation. Henry suggested, therefore, that substance P may be involved specifically in afferent units associated with pain sensation. Using immunohistochemical techniques for substance P, our finding of extensive staining of the nerve plexus in lamina V below the lesion (Fig. 3b and c) shows the presence of a network ofvaricosities in the nerve endings in an area classically known to be concerned with noxious transmission. These networks were never observed in the sections above the lesion. This finding demonstrates a build-up of substance P below the lesion, and suggests its rostral direction by axoplasmic flow in the spinal cord. H6kfelt also observed that substance P-containing fibers in peripheral afferent nerves are unmyelinated, and that in the skin these fibers terminate in free nerve endings usually associated with pain transmission ~ . Cerebral cortical neurons in cats and rats were much less readily excited than spinal interneurons by substance P. Cuneate neurons in the cat produced a slow excitation, beginning after a delay of 10-30 sec when substance P was applied iontophoretically 1~. These neurons reached a peak approximately 30 sec later and, after the end of application, the excitation declined over 1 rain or longer. It was concluded that the slow time course of substance P action was imcompatible with a role as the main excitatory transmitter of primary afferent terminals, but its strong excitatory action might have functional significance as sensitizer or modulator over a long period. Recently, Pickel et a1.16 using immunocytochemical techniques, reported that within axon terminals substance P appeared to be associated with one type of organelle, a large, round vesicle 60-80 nm in diameter. In addition, there were also present unlabeled, small vesicles in the same axon terminal. These small vesicles are generally accepted as being the storage sites of most neurotransmitters~ This finding further supports the suggestion that substance P may act as a modulator rather than the neurotransmitter initiating synaptic events. Our data concur with those of other investigators, suggesting a pathway for substance P starting from the dorsal root ganglion via primary afferent fibers toward their terminals in the dorsal horn of the spinal cord. It further extends the work by demonstrating that substance P accumulates in the dorsolateral part of the dorsal horn below the level of the lesion, indicating an upward flow of this peptide in the spinal cord in contrast to downward movement from brain stem of monoamine transmitters
513 by axoplasmic flow '~,t4. It was of interest to note the presence of some fibers stained for substance P in the ventral horn. The a m o u n t of i m m u n o r e a c t i v e p r o d u c t appeared to remain c o n s t a n t above a n d below transection, suggesting that the substance P fibers in the ventral horn are involved in spinal segmental transmission. In contrast, the fibers in the dorsal h o r n were f o u n d to be ascending. A l t h o u g h our data indicate an anterograde direction of substance P in the spinal cord, they do n o t elucidate whether it arises in the collaterals of primary afferent fibers or in second-order fibers. ACKNOWLEDGEMENTS Supported
in part
by the
Edmond
A.
G u g g e n h e i m Clinical Research
E n d o w m e n t a n d RSA G r a n t s 13-P-57975/2-01 a n d 13-P-55868/2. REFERENCES 1 Brownstein, M. J., Mroz, E. A., Kizer, J. S., Palkovits, M. and Leeman, S. E., Regional distribution of substance P in the brain of the rat, Brain Research, 116 (1976) 229-305. 2 Chang, M. M. and Leeman, S. E., Isolation ofa sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P, J. biol. Chem., 245 (1970) 4784 4790. 3 Chang, M. M., Leeman, S. E. and Niall, H. D., Amino acid sequence of substance P, Nature new Biol., 232 (1971) 86-87. 4 Cuello, A. C., Polak, J. M. and Pearse, A. G. E., Substance P: a naturally occurring transmitter in human spinal cord, The Lancet, November 13 (1976) 1054-1056. 5 Dahlstr6m, A., Axoplasmic transport, Phil. Trans. B, 261 (1971) 325-358. 6 Henry, J. L.•Effects•fsubstanceP•nfuncti•na••yidenti•edunitsincatspina•c•rd•BrainResearch• 114 (1976) 439-451. 7 Henry, J. L., Krnjevid, K. and Morris, M. E., Substance P and spinal neurones, Canad. J. Physiol. Pharmaeol., 53 (1975) 423-432. 8 H6kfelt, T., Kellerth, J. O., Nillson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 9 H6kfelt, T., Kellerth, J. O., Nillson, G. and Pernow, B., Substance P: localization in the central nervous system and some primary sensory neurons, Science, 190 (1975) 889-890. 10 Konishi, S. and Otsuka, M., The effects of substance P and other peptides on spinal neurons of the frog, Brain Research, 65 (1974) 397-410. 1I Konishi, S. and Otsuka, M. ,Excitatory action of hypothalamic substance P on spinal motoneurons of newborn rats, Nature (Lond.), 252 (1975) 734-735. 12 Krnjevid, K. and Morris, M. E., An excitatory action of substance P on cuneate neurons, Canad. J. Physiol. Pharmacol., 52 (1974) 736-744. 13 Naftchi, N. E., Abrahams, S. J., St. Paul, H. and Lowman, E. W., Localization of substance P in the spinal cord of paraplegic rats, Trans. Amer. Soc. Neurochem., 8 (1977) 271. 14 Naftchi, N. E., Demeny, M., Kertesz, A., Viau, A. T. and Lowman, E. W., Effect of spinal cord transection on mammalian biogenic amines, c-AMP, and tyrosine hydroxylase activity in CNS, adrenals and heart, Trans. Amer. Soc. Neurochem., 5 (1974) 80. 15 Otsuka, M., Konishi, S. and Takahashi, T., Hypothalamic substance P as a candidate for transmitter of primary afferent neurons, Fed. Proc., 34 (I 975) 1922-I 928. 16 Pickel, V. M., Reis, D. J. and Leeman, S. E., Ultrastructural localization of substance P in neurons of rat spinal cord, Brain Research, 122 (1977) 534-540. 17 Pickel, V. M., Tong, H. J., Field, P. M., Becker, C. G. and Reis, D. J., Cellular localization of tyrosine hydroxylase by immunohistochemistry, J. Itistochem. Cytochem., 23 (1975) 1 12. 18 Powell, D., Leeman, S., Tregear, G. W., Niall, H. D. and Potts, J. T., Radioimmunoassay for substance P, Nature new Biol., 241 (1973) 252 254. 19 Rexed, B., A cytoarchitectonic atlas of the spinal cord in the cat, J. comp. Neurol., 100 (1954) 297379. 20 Sternberger, L., Immunocytochemistry, Prentice-Hall, Englewood Cliffs, N. J., 1974. 21 Von Euler, U. S. and Gaddum, J. H., An unidentified depressor substance in certain tissue extracts, J. Physiol. (Lond.), 72 (1931) 74-87.
507
L O C A L I Z A T I O N A N D C H A N G E S OF SUBSTANCE P I N S P I N A L C O R D OF P A R A P L E G I C CATS
N. E. NAFTCHI, S. J. A B R A H A M S , H. M. ST. PAUL, E. W. LOWMAN and W. SCHLOSSER
Laboratory of Biochemical Pharmacology, lnstHute of Rehabilitation Medicine, New York University Medical Center, 400 East 34th Street, New York, N. Y. 10016and (W.S.) Hoffmann-La Roche, Research Division, Nutley, N.J. 07110 (U.S.A.)
(Accepted January 18th, 1978)
SUMMARY Female cats were transected at the T12 region of the spinal cord and sacrificed by perfusion through the aorta after 5 days, 2, 5, 8 or 12 weeks. Tissue from above and below the lesion was postfixed, dehydrated and embedded in paraffin. 5 # m sections were incubated for substance P immunoreactivity by the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Spinal cord sections of normal cats showed a small amount of peroxidase-positive staining in the dorsal horn in laminae I, II and III of Rexed. Starting at 5 days after transection, there was a sharp increase in the amount of substance P-staining below the lesion in the form of punctate bodies and long varicosities. Above the lesion in the region of the substantia gelatinosa the number of stained bodies was fewer and the intensity of staining around the dorsal horns was less than that in sections obtained from below the lesion. This pattern was repeated when sections from animals sacrificed at later times were stained for substance P. The results confirm the presence of substance P in the spinal cord nerve tracts. Its accumulation in the dorsal horn below the transection lends credence to its role as a transmitter or modulator of afferent neuronal activity, and suggests its rostral direction by axoplasmic flow.
INTRODUCTION In 1931, Von Euler and Gaddum extracted a factor from equine brain and intestine which they called substance p21. It was found to possess a vasodilatory effect and stimulate smooth muscle. It was not until 1970 when Chang and Leeman 2 isolated a sialogogic peptide from bovine hypothalamus that it was finally purified, characterized as substance P, synthesized, and its regional distribution studied by radio-
508 immunoassay and immunofluorescence techniquesa, z,~8. Leeman and coworkers have shown substance P to be an undecapeptide 2. It has an uneven distribution in the CNS. Its highest concentrations are found in the substantia nigra, hypothalamus, pineal gland and the dorsal gray matter of the spinal cord1,'4, 9. When applied directly to single neurons, such as the motor neuron of the ventral horn of the spinal cord, it provokes a slow, long-lasting excitatory response tS. It has been localized by immunofluorescence in the substantia gelatinosa of the dorsal horn of human spinal cord by Cuello et al. 4, and in the same area of the cat and the rat by H6kfelt ct al.S,% The immunoreactive fibers seem to represent thin unmyelinated fibers classically known to carry thermal and pain stimuli. The latter finding suggests that substance P is the first chemical signal for exteroceptive perception in the human spinal cord '1. In this study. we have localized substance P in the spinal cords of cats by the peroxidase-antiperoxidase (PAP) immunocytochemical technique, and have followed its distribution after the transection of the spinal cord. A preliminary report of our findings has been published 13. MATERIALS AND METHODS
Preparation of animals Female cats weighing between 2.1 and 3.2 kg were selected and anesthetized with 30 mg/kg ketamine, i.m. A dorsal laminectomy was performed and the spinal cord was exposed and transected at T12. Small pieces of sterile gelfoam were placed between the cut ends of the cord in the cats which immediately stopped any bleeding that had occurred. The gelfoam was left in place and the animals were sutured. Animals were not fed or watered for one day following surgery, after which they were allowed to eat or drink ad libitum, and their bladders were expressed 3 times daily.
Tissue preparation At varying time intervals after transection, the animals were anesthetized with Nembutal and sacrificed by perfusion through the ascending aorta with 4 ~ paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 for 10 min in. Sections approximately 2 cm long were removed from above and below the lesion in the spinal cord. These pieces were immediately immersed in picric acid-formaldehyde fixative for about 6 h, after which time they were cut into smaller pieces. The tissues were then rinsed in phosphate-buffered saline from 2 h to overnight, dehydrated through a graded series of ethanol solutions, put through several changes of xylene and paraffin, and finally embedded in paraffin. Sections were cut at a thickness of 4-5 # m and affixed to albumin-coated glass slides. Once prepared, these slides could be incubated for immunoreactivity at any future time.
Antisera Rabbit antiserum to substance P was generously given to us b y Dr. Susan Leeman. Prior to incubation of the tissue, the antiserum was diluted 1:100 with 0.5 M
509 Tris.saline (pH 7.6) and incubated for 2 h at 37 °C with rat liver acetone powder (20 mg liver powder/ml of diluted serum). This was stored overnight at 4 °C and filtered the next morning through a 0.2 #m millipore filter. It was then further diluted with 0.5 M Tris.saline. Final dilutions used in this series of experiments ranged from 1:400 to 1 : 1000.
Immunohistochemical staining The peroxidase-anti-peroxidase immunohistochemical method as first described by Sternberger 2° and modified by Pickel et al. iv was performed on 5/~m tissue sections for localization of substance P in the cat tissue. Non-specific protein binding was reduced by incubating the sections with 3 goat serum in Tris.saline before they were incubated with anti-substance P antiserum for 1 h. Goat anti-rabbit immunoglobulin (Miles Labs) was applied for 30 min after two brief rinses with Tris.saline. Following two more rinses, the PAP reagent (Dako Accurate Chemicals) was applied in 1:25 dilution for 30 min. The slides were then incubated for 15-30 min with 0 . 0 5 ~ 3,3'-diaminobenzidine (Sigma) and 0.01~,i hydrogen peroxide solution in Tris buffer (pH 7.6). A brown precipitate was formed characteristic of polymerized diaminobenzidine. After several washes in distilled water, the slides were dehydrated and mounted with coverslips. Sham-operated animals served as controls. Tissues from these animals were processed in the same manner as above. In order to control for immunohistochemical specificity, normal rabbit serum was routinely substituted for specific rabbit antibody to substance P in all experiments. The sections were examined with a Jena light microscope at magnifications of 25-630 ×. RESULTS Tissue from sham-operated cats (Fig. la and b) displayed a small amount of peroxidase-positive staining which appeared as a band of nerve terminals in the dorsal horn at the junction between the white and grey matters, the substantia gelatinosa which corresponds to laminae II and Ill of Rexed 19. Five days after transection, there was a sharp increase in the amount of substance P below the lesion which had outlined both dorsal horns and bridged the two together. Above the lesion, in the region of the substantia gelatinosa, the number of punctate bodies was fewer, and the intensity of staining around the dorsal horns was less than that in sections obtained from below the lesion. This pattern was repeated when sections from animals sacrificed 1, 2, 5, 8 and 12 weeks after transection were stained for substance P. Specific staining in sections cut from above the lesion (Fig. 2a and 3a), was little compared with the great amount of staining below (Figs. 2b and 3b). In addition to punctate bodies, long fibers and varicosities were present in sections taken from below the lesion (Fig. 2c), and a network of fibers located centrally within each dorsal horn from cats sacrificed 12 weeks after transection was positively stained (Figs. 3b and c). A small amount of immunoreactive product was often seen in the
Fig. I. a: section from TI 1 12 region of the spinal cord of a s h a m - o p e r a t e d cat. Arro~,~ noint to in~m u n o r e a c t i v e s u b s t a n c e P (SP) specific bodies in dorsal horn (area o f s u b s t a n t i a gelatinosa, SGt, 200. (Bar : 100/tin in all figures), b: phase contrast micrograph of the s a m e dorsal h o r n reginri seen in ~. T h e section was i n c u b a t e d for SP immunoreaclivity. Note the presence of a dark ring in the SG regioH, indicating the area of immunoreactivity. 200. c : phase contrast m i c r o g r a p h of the same dorsal h o r n region seen in a. T h e section was incubated with n o r m a l rabbii serum. C o m p a r e d t{~ b. ~,he ~bsence ~f a n y SP-specific staining is evident. 200,
Fig. 2. a: field s h o w i n g dorsal h o r n s abow~ the lesion from a cat transected 5 weeks belk)re perfusion. SG s h o w s slight staining, ;~ 125. b: tissue from the s a m e cat as in a sectioned below the lesion. Note the intense reaction p r o d u c t delineating both dorsal horns. :, 100. c: this is a detail o f the SG seen in b. A r r o w s point to darkly stained S P - i m m u n o r e a c t i v e punctate bodies a n d a n intense band of bead-like varicosities delineating the entire SG, ~ 250. d: section o f the ventral h o r n from below the lesion of a cat transected 5 weeks before sacrifice. Ventral h o r n cells (V) are present. A r r o w s point to possible i m m u n o positive bodies that are scattered t h r o u g h o u t . 250,
511
Fig. 3. a: section from above the lesion of a cat transected 12 weeks before sacrifice. Note sparse staining surrounding the dorsal horn in the SG. x 200. b: tissue from the same cat as in a, sectioned below the lesion. The staining of the band of nerve terminals in the SG is much more intense in this section than that in the section above the lesion. Note the appearance of the nerve plexus (arrow) near the center (Lamina V) of the dorsal horn. x 160. c: a detail of the nerve plexus from b. Immunoreactive substance appears in bead-like, fibrillar varicosities which may be nerve terminals originating from dorsal roots and entering the spinal cord. RBC, red blood cells present in small capillaries. ~. 400. ventral horn in the f o r m o f small, dense p u n c t a t e bodies scattered r a n d o m l y t h r o u g h o u t the horn (Fig. 2d). It was difficult to assess whether the a m o u n t o f staining changed with time either a b o v e or below the lesion in the ventral horn. S u b s t i t u t i o n o f n o r m a l serum for i m m u n e serum as a control showed no staining in the grey m a t t e r o f the spinal cord, with the exception o f red b l o o d cells, which contain e n d o g e n o u s peroxidase (Fig. lc). In some specimens, i m m u n o r e a c t i v e staining did a p p e a r in the white matter, which could be a t t r i b u t e d to some degree o f nonspecific affinity o f the r a b b i t serum for the m e m b r a n e in the region where the myelin had leached out, since the same type of light-brown b a c k g r o u n d staining a p p e a r e d in control sections where n o r m a l r a b b i t serum was substituted for the specific antiserum. There were, however, some small, d a r k punctate bodies present in the myelinated axons o f the white m a t t e r in the ventrolateral and d o r s o l a t e r a l parts o f the spinal cord. This m a y suggest r o s t r a l a x o n a l flow o f substance P within m y e l i n a t e d fibers, possibly belonging to lateral s p i n o t h a l a m i c tracts or to shorter tracts concerned with segmental transmission. DISCUSSION The a b u n d a n c e o f substance P - i m m u n o r e a c t i v e nerve terminals in the substantia gelatinosa suggests t h a t substance P is contained in the p r i m a r y afferent fibers which t e r m i n a t e in the d o r s a l horn. O t s u k a et al. 15 ligated the d o r s a l r o o t s o f the cat and f o u n d that substance P a c c u m u l a t e d on the ganglion side o f the ligature, but its level on the central side decreased. The finding o f substance P in these afferent fibers suggested a role in neurotransmission. W o r k o f Konishi a n d O t s u k a 10 on the isolated
512 frog spinal cord preparation has shown substance P to be about 200 times more active on a molar basis than L-glutamate in depolarizing spinal motor neurons. This depolarizing action persisted after synaptic transmission was blocked by Ca'-'~-deficient Ringer's solution or by tetrodotoxin. They concluded, therefore, that substance P exerted a transsynaptic action on motor neurons, and was probably a candidate for the excitatory transmitter of primary sensory neurons 10,11. Iontophoretic application of substance P to spinal neurons by Henry6, 7 produced a strong but slow and prolonged excitatory action on nearly half the neurons tested in the lumbar spinal cord of the cat. It is noteworthy that all units excited by substance P were also excited by noxious thermal stimulation of the skin. The highest number of units excited by substance P was found in lamina VI and the lowest in lamina IV. In two cases, treatment with substance P led to a response to noxious heat by units which had previously been unresponsive to thermal stimulation. Henry suggested, therefore, that substance P may be involved specifically in afferent units associated with pain sensation. Using immunohistochemical techniques for substance P, our finding of extensive staining of the nerve plexus in lamina V below the lesion (Fig. 3b and c) shows the presence of a network ofvaricosities in the nerve endings in an area classically known to be concerned with noxious transmission. These networks were never observed in the sections above the lesion. This finding demonstrates a build-up of substance P below the lesion, and suggests its rostral direction by axoplasmic flow in the spinal cord. H6kfelt also observed that substance P-containing fibers in peripheral afferent nerves are unmyelinated, and that in the skin these fibers terminate in free nerve endings usually associated with pain transmission ~ . Cerebral cortical neurons in cats and rats were much less readily excited than spinal interneurons by substance P. Cuneate neurons in the cat produced a slow excitation, beginning after a delay of 10-30 sec when substance P was applied iontophoretically 1~. These neurons reached a peak approximately 30 sec later and, after the end of application, the excitation declined over 1 rain or longer. It was concluded that the slow time course of substance P action was imcompatible with a role as the main excitatory transmitter of primary afferent terminals, but its strong excitatory action might have functional significance as sensitizer or modulator over a long period. Recently, Pickel et a1.16 using immunocytochemical techniques, reported that within axon terminals substance P appeared to be associated with one type of organelle, a large, round vesicle 60-80 nm in diameter. In addition, there were also present unlabeled, small vesicles in the same axon terminal. These small vesicles are generally accepted as being the storage sites of most neurotransmitters~ This finding further supports the suggestion that substance P may act as a modulator rather than the neurotransmitter initiating synaptic events. Our data concur with those of other investigators, suggesting a pathway for substance P starting from the dorsal root ganglion via primary afferent fibers toward their terminals in the dorsal horn of the spinal cord. It further extends the work by demonstrating that substance P accumulates in the dorsolateral part of the dorsal horn below the level of the lesion, indicating an upward flow of this peptide in the spinal cord in contrast to downward movement from brain stem of monoamine transmitters
513 by axoplasmic flow '~,t4. It was of interest to note the presence of some fibers stained for substance P in the ventral horn. The a m o u n t of i m m u n o r e a c t i v e p r o d u c t appeared to remain c o n s t a n t above a n d below transection, suggesting that the substance P fibers in the ventral horn are involved in spinal segmental transmission. In contrast, the fibers in the dorsal h o r n were f o u n d to be ascending. A l t h o u g h our data indicate an anterograde direction of substance P in the spinal cord, they do n o t elucidate whether it arises in the collaterals of primary afferent fibers or in second-order fibers. ACKNOWLEDGEMENTS Supported
in part
by the
Edmond
A.
G u g g e n h e i m Clinical Research
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