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Reduced substance P in hereditary sensory neuropathy in themf rat
147
Brain Research, 263 (1983) 147-150 Elsevier Biomedical Press
Reduced substance P in hereditary sensory neuropathy in the mf rat F. SCARAVILLI
Department of Neuropathology, Institute of Neurology, The National Hospital, Queen Square, London (U.K.) (Accepted November 9th, 1982)
Key words: neurotransmitters - substance P - sensory neuropathy - neurological mutants - rat - mfmutant
Mutilated foot (mf) is a mutant rat with an autosomal recessive sensory neuropathy. Affected animals become ataxic and their feet become mutilated. Morphological and quantitative studies have shown a reduced number of sensory ganglion cells and of cells of secondary sensory neurons. No degeneration was seen in the peripheral nervous system. Substance P (SP) is an undecapeptide which is thought to be involved in transmission of nociceptive information. Since mfrats show, in addition to ataxia, a decreased response to painful stimuli, SP immunoreactivity was examined. The density of SP staining was decreased at all levels of the spinal cord, mainly at cervical and lumbar levels and only in areas related to sensory pathways. In other areas of the spinal cord and in the substantia gelatinosa of the trigeminal tract, no reduction of SP staining was observed. The results further support the relationship between SP and transmission of pain stimuli. There are also many similarities between the appearances in mfrats and those in animals in which sensory afferents from dorsal root ganglia had been impaired with various methods, particularly in those treated from birth with capsaicin.
Substance P (SP), first identified by von Euler and G a d d u m 5, is an undecapeptide 4 that has a widespread distribution in the CNS, where it is particularly concentrated in the substantia nigra and dorsal horn of the spinal cord 3.It. SP is considered to be involved in transmission of nociceptive impulses at synapses of primary sensory neurons 7.13. Experimental lesions of the primary sensory neuron 2.9.2° induce depletion of SP from the substantia gelatinosa of the spinal cord. A reduction of SP-like immunoreactivity has been found in the CSF of patients suffering from peripheral neuropathies with values that tended to correlate with sensory rather than motor impairment 14. In familial dysautonomia (the RileyDay syndrome), in which a reduced number of primary sensory neurons is associated with insensitivity to pain and temperature ~5, there is much reduced SP-immunoreactivity in the substantia gelatinosa of the medulla and spinal cord t6. In 1981, Jacobs et al.8 described a new neurological mutant in the rat, mutilated foot (mJ). This disease is inherited as an autosomal recessive. Affected animals become ataxic and their 0006-8993/83/0000-0000/$03.000 1983 Elsevier Biomedical Press
feet, generally of the hind limbs, become mutilated. Morphological and quantitative studies showed severe reductions in the number of sensory ganglion cells and fibers, including unmyelinated fibers and a reduction in size of the dorsal horn of spinal cord. The CNS pathology extends to the secondary sensory neurons that are extremely reduced in number ~s. The changes in this mutant are comparable with those in various human hereditary sensory neuropathies, one of which is recessively inherited, non-progressive '9, and does not show signs of degeneration in the peripheral nervous system t2. It was concluded that the mfrat could be a useful model for the study of hereditary sensory neuropathies in man. Since affected animals, in addition to ataxia, show a decreased response to painful stimuli, SP immunoreactivity related to the sensory pathways was examined. Five mfrats of both sexes, aged 4-15 months, and an equal number of normal littermates, matched for sex and body weight, were perfused under pentobarbitone anesthesia with 0.4% pbenzoquinone in phosphate buffer at pH 7.2. Transverse sections of the upper medulla and of
148 ii!~i¸iii
Fig. 1. Peroxidase-antiperoxidase SP staining in the dorsal horn L5 segment of mf(A) and control (B) rats. In the mutant the area of the substantia gelatinosa and the intensity of its SP staining are severely reduced compared with the control. x 60.
the spinal cord at C6, T7 and L5 levels were cut and processed for the identification of SP according to the method described by Barbut et al.2. In the control animals, the distribution of SP at the levels examined was comparable to that previously described ~1in rats. In the medulla of the mf rat the size of the marginal layer and substantia gelatinosa of the trigeminal tract containing SP is the same as in controls and the density of the staining did not appear to be reduced. SP-positive axons were
also found in the spinal tract of the trigeminal nerve. At level C6, both the area and the density of SP staining were considerably less than in controis. This reduction affects all the regions of the dorsal horn, but is mostly evident in the middle and lateral third of the laminae. The dorso-lateral funiculus also shows smaller amounts of SP. The reduction in SP-immunoreactivity at L5 is very severe (Fig. IA, B) and more evident than at higher levels. The zone of Lissauer and the dorso-lateral funiculus, strongly reactive in control rats, are extremely faint in mfrats and positive granules in laminae I and II are sparse and smaller than in controls. At thoracic level there is little difference in the size of the dorsal horn in mfcompared with control rats and the density of SP staining is only slightly reduced in affected animals. At these various levels of the spinal cord, the intensity of SP reactivity in other areas not related to sensory pathways was also examined. The fine, delicate network of fibers surrounding motor neurons in lamina IX (Fig. 2A), the plexus of the intermedio-lateral column (Fig. 2B) and the band of thin SP-positive fibers directed towards the central canal do not show any reduction in density of staining in affected rats. The plexus around the central canal with the area of strong SP density ventral to it has the same intensity of staining in the two groups of animals (Fig. 2C). The results of the present study show that in mfrats there is an abnormally low SP immunoreactivity in areas of the spinal cord receiving inputs from the dorsal root ganglia in keeping with the low numbers of ganglion cells at cervical and lumbar levels8 and with the hypothesis that SP is involved in the transmission of stimuli related to pain. The more severe involvement of the substantia gelatinosa of the cervical and lumbar enlargements compared with that at thoracic level in mfrats could be explained by the more severe loss of sensory ganglion cells at these two levels than in the thoracic region. The presence of apparently normal amounts of SP in axons of the trigeminal pathway is also in agreement with the finding (Jacobs, personal communication) that the trigeminal ganglion is less severely affected
149
Fig. 2. Peroxidase-antiperoxidase SP staining of the mfrat. A: lamina IX at lumbar level L5. B: intermedio-lateral column at T7. C : gray matter around the central canal. In all these areas the intensity of SP staining is the same as in controls, x 375, 600 and 480, respectively.
than spinal ganglia and with the observation that there is no apparent clinical abnormality at this level. The reason for the difference between cranial and spinal ganglia is not clear. It might reflect a different embryological origin 2~, as suggested by Pearson et al. 16. Morphological appearances of the dorsal horns of the cervical and lumbar enlargements in mfrats resemble those seen in animals that were subject to dorsal root
section L2°, peripheral nerve i n j u r f and treatment with capsaicin 9. In these conditions, as in the mfrat, the dorsal horns are smaller and their shape is rounder than in control animals. Furthermore, the appearances in the rnf rat have close similarities with those found in animals treated with capsaicin during the neonatal period 6, in both cases the levels of SP being irreversibly reduced in the dorsal horns while they are
150 n o r m a l in the v e n t r a l h o r n s . I n c a p s a i c i n - t r e a t e d a n i m a l s , h o w e v e r , t h e r e is i n v o l v e m e n t o f the s p i n a l t r i g e m i n a l tract t h a t is s p a r e d in o u r m o d el. R e c e n t l y the effect o f n e r v e g r o w t h f a c t o r ( N G F ) o n SP at v a r i o u s levels has b e e n studied 1°.17. T h e s e results c o u l d be r e l e v a n t for the u n d e r s t a n d i n g o f the p a t h o g e n e s i s o f the disease in the m f r a t since t r e a t m e n t with N G F increases SP in g a n g l i a a n d s p i n a l d o r s a l h o r n s while it has n o s i g n i f i c a n t effect o n v e n t r a l h o r n s . The observations reported here indicate that
in the i n h e r i t e d s e n s o r y n e u r o p a t h y in the rat t h e r e is a r e d u c t i o n o f S P - i m m u n o r e a c t i v i t y in sensory pathways.
1 Barber, R. P., Vaughn, J. E., Slemmon, J. R., Salvaterra, P. M., Roberts, E. and Leeman, S. E., The origin, distribution and synaptic relationships of substance P axons in rat spinal cord,./, comp. Neurol., 184 (1979) 331-352. 2 Barbut, D., Polak, J. M. and Wall, P. D., Substance P in spinal cord dorsal horn decreases following peripheral nerve injury, Brain Research, 205 (198 !) 289-298. 3 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) 299305. 4 Chang, M. M., Leeman, S. E. and Nial, H. D., Aminoacid sequence of substance P, Nature New Biol., 232 (1971) 86-87. 5 Euler, U. S. von and Gaddum, J. H., An unidentified depressor substance in certain tissue extracts, J. Physiol. (Lond.), 72 (1931) 74-87. 6 Helke, C. J., DiMicco, J. A., Jacobowitz, D. M. and Kopin, I.J., Effects of capsaicin administration to neonatal rats on the substance P content of discrete CNS regions, Brain Research, 222 (1981) 428-431. 7 Henry, J. L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 439-451. 8 Jacobs,.J.M., Scaravilli, F., Duchen, L. W. and Mertin, J., A n~w neurological rat mutant 'mutilated foot', J. Anat., 132 (1981) 525-543. 9 JesseU, T. M., Iversen, L. L. and Cuello, A. C., Capsaicininduced depletion of substance P from primary sensory neurons, Brain Research, 152 (1978) 183-188. 10 Kessler, J. A. and Black, I. B., Nerve growth factor stimulates development of substance P in the embryonic spinal cord, Brain Research, 208 (198 !) 135 145. 11 Ljungdahl, A., H6kfelt, T. and Nilsson, G., Distribution of substance P-like immunoreactivity in the central ner-
vous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-943. 12 Murray, T. J., Congenital sensory neuropathy, Brain, 96 (1973) 387-394. 13 Nicoll, R. A., Schenker, C. and Leeman, S. E., Substance P as a transmitter candidate, Ann. Rev. Neurosci., 3 (1980) 227-268. 14 Nutt, J. G., Mroz, E. A., Leeman, S. E., Williams, A. C., Engel, W. K. and Chase, T. N., Substance P in human cerebrospinal fluid: reduction in peripheral neuropathy and autonomic dysfunction, Neurology, 30 (1980) 1280 1285. 15 Pearson, J., Familial dysautonomia (a brief review), J. A utonom. Nerv. Syst., 1 (1979) 119-126. 16 Pearson, J., Brandeis, L. and Cuello, A. C., Depletion of substance P-containing axons in substantia gelatinosa of patients with diminished pain sensitivity, Nature (Lond.), 295 (1982) 61-63. 17 Ross, M., L6fstrandh, S., Gorin, P. D., Johnson, E. M. and Schwartz, J. P., Use of an experimental autoimmune model to define nerve growth factor dependency of peripheral and central substance P-containing neurons in the rat, J. Neurosci., 1 (1981) 1304-1311. 18 Scaravilli, F. and Jacobs, J. M., Quantitative studies of motor, spinocerebellar and secondary sensory neurons in the mutilated foot mutant rat, Neuroscience, 6 (1981) 1663-1674. 19 Spillane, J. D. and Wells, C. E. C., Acrodystrophic Neuropathy, Oxford University Press, London, 1969. 20 Tessler, A., Glazer, E., Artymyshyn, R., Murray, M. and Goldberger, M. E., Recovery of substance P in the cat spinal cord after unilateral lumbosacral deafferentation, Brain Research, 191 (1980) 459-470. 21 Theiler, K., The House Mouse, Springer-Verlag, Berlin, 1972.
W e s h o u l d like to t h a n k Miss M a r g a r e t W h i t e for the h i s t o c h e m i c a l p r e p a r a t i o n s , Mr. J. A. Mills f o r h e l p with the p h o t o g r a p h y , Mrs. M a r y H a l l i d a y for the t y p i n g , a n d Dr. P. E m s o n f o r the gift o f the a n t i b o d y . This w o r k was s u p p o r t ed b y g r a n t s f r o m the N a t i o n a l F u n d f o r M e d i cal R e s e a r c h into C r i p p l i n g D i s e a s e s a n d the Medical Research Council.
Brain Research, 263 (1983) 147-150 Elsevier Biomedical Press
Reduced substance P in hereditary sensory neuropathy in the mf rat F. SCARAVILLI
Department of Neuropathology, Institute of Neurology, The National Hospital, Queen Square, London (U.K.) (Accepted November 9th, 1982)
Key words: neurotransmitters - substance P - sensory neuropathy - neurological mutants - rat - mfmutant
Mutilated foot (mf) is a mutant rat with an autosomal recessive sensory neuropathy. Affected animals become ataxic and their feet become mutilated. Morphological and quantitative studies have shown a reduced number of sensory ganglion cells and of cells of secondary sensory neurons. No degeneration was seen in the peripheral nervous system. Substance P (SP) is an undecapeptide which is thought to be involved in transmission of nociceptive information. Since mfrats show, in addition to ataxia, a decreased response to painful stimuli, SP immunoreactivity was examined. The density of SP staining was decreased at all levels of the spinal cord, mainly at cervical and lumbar levels and only in areas related to sensory pathways. In other areas of the spinal cord and in the substantia gelatinosa of the trigeminal tract, no reduction of SP staining was observed. The results further support the relationship between SP and transmission of pain stimuli. There are also many similarities between the appearances in mfrats and those in animals in which sensory afferents from dorsal root ganglia had been impaired with various methods, particularly in those treated from birth with capsaicin.
Substance P (SP), first identified by von Euler and G a d d u m 5, is an undecapeptide 4 that has a widespread distribution in the CNS, where it is particularly concentrated in the substantia nigra and dorsal horn of the spinal cord 3.It. SP is considered to be involved in transmission of nociceptive impulses at synapses of primary sensory neurons 7.13. Experimental lesions of the primary sensory neuron 2.9.2° induce depletion of SP from the substantia gelatinosa of the spinal cord. A reduction of SP-like immunoreactivity has been found in the CSF of patients suffering from peripheral neuropathies with values that tended to correlate with sensory rather than motor impairment 14. In familial dysautonomia (the RileyDay syndrome), in which a reduced number of primary sensory neurons is associated with insensitivity to pain and temperature ~5, there is much reduced SP-immunoreactivity in the substantia gelatinosa of the medulla and spinal cord t6. In 1981, Jacobs et al.8 described a new neurological mutant in the rat, mutilated foot (mJ). This disease is inherited as an autosomal recessive. Affected animals become ataxic and their 0006-8993/83/0000-0000/$03.000 1983 Elsevier Biomedical Press
feet, generally of the hind limbs, become mutilated. Morphological and quantitative studies showed severe reductions in the number of sensory ganglion cells and fibers, including unmyelinated fibers and a reduction in size of the dorsal horn of spinal cord. The CNS pathology extends to the secondary sensory neurons that are extremely reduced in number ~s. The changes in this mutant are comparable with those in various human hereditary sensory neuropathies, one of which is recessively inherited, non-progressive '9, and does not show signs of degeneration in the peripheral nervous system t2. It was concluded that the mfrat could be a useful model for the study of hereditary sensory neuropathies in man. Since affected animals, in addition to ataxia, show a decreased response to painful stimuli, SP immunoreactivity related to the sensory pathways was examined. Five mfrats of both sexes, aged 4-15 months, and an equal number of normal littermates, matched for sex and body weight, were perfused under pentobarbitone anesthesia with 0.4% pbenzoquinone in phosphate buffer at pH 7.2. Transverse sections of the upper medulla and of
148 ii!~i¸iii
Fig. 1. Peroxidase-antiperoxidase SP staining in the dorsal horn L5 segment of mf(A) and control (B) rats. In the mutant the area of the substantia gelatinosa and the intensity of its SP staining are severely reduced compared with the control. x 60.
the spinal cord at C6, T7 and L5 levels were cut and processed for the identification of SP according to the method described by Barbut et al.2. In the control animals, the distribution of SP at the levels examined was comparable to that previously described ~1in rats. In the medulla of the mf rat the size of the marginal layer and substantia gelatinosa of the trigeminal tract containing SP is the same as in controls and the density of the staining did not appear to be reduced. SP-positive axons were
also found in the spinal tract of the trigeminal nerve. At level C6, both the area and the density of SP staining were considerably less than in controis. This reduction affects all the regions of the dorsal horn, but is mostly evident in the middle and lateral third of the laminae. The dorso-lateral funiculus also shows smaller amounts of SP. The reduction in SP-immunoreactivity at L5 is very severe (Fig. IA, B) and more evident than at higher levels. The zone of Lissauer and the dorso-lateral funiculus, strongly reactive in control rats, are extremely faint in mfrats and positive granules in laminae I and II are sparse and smaller than in controls. At thoracic level there is little difference in the size of the dorsal horn in mfcompared with control rats and the density of SP staining is only slightly reduced in affected animals. At these various levels of the spinal cord, the intensity of SP reactivity in other areas not related to sensory pathways was also examined. The fine, delicate network of fibers surrounding motor neurons in lamina IX (Fig. 2A), the plexus of the intermedio-lateral column (Fig. 2B) and the band of thin SP-positive fibers directed towards the central canal do not show any reduction in density of staining in affected rats. The plexus around the central canal with the area of strong SP density ventral to it has the same intensity of staining in the two groups of animals (Fig. 2C). The results of the present study show that in mfrats there is an abnormally low SP immunoreactivity in areas of the spinal cord receiving inputs from the dorsal root ganglia in keeping with the low numbers of ganglion cells at cervical and lumbar levels8 and with the hypothesis that SP is involved in the transmission of stimuli related to pain. The more severe involvement of the substantia gelatinosa of the cervical and lumbar enlargements compared with that at thoracic level in mfrats could be explained by the more severe loss of sensory ganglion cells at these two levels than in the thoracic region. The presence of apparently normal amounts of SP in axons of the trigeminal pathway is also in agreement with the finding (Jacobs, personal communication) that the trigeminal ganglion is less severely affected
149
Fig. 2. Peroxidase-antiperoxidase SP staining of the mfrat. A: lamina IX at lumbar level L5. B: intermedio-lateral column at T7. C : gray matter around the central canal. In all these areas the intensity of SP staining is the same as in controls, x 375, 600 and 480, respectively.
than spinal ganglia and with the observation that there is no apparent clinical abnormality at this level. The reason for the difference between cranial and spinal ganglia is not clear. It might reflect a different embryological origin 2~, as suggested by Pearson et al. 16. Morphological appearances of the dorsal horns of the cervical and lumbar enlargements in mfrats resemble those seen in animals that were subject to dorsal root
section L2°, peripheral nerve i n j u r f and treatment with capsaicin 9. In these conditions, as in the mfrat, the dorsal horns are smaller and their shape is rounder than in control animals. Furthermore, the appearances in the rnf rat have close similarities with those found in animals treated with capsaicin during the neonatal period 6, in both cases the levels of SP being irreversibly reduced in the dorsal horns while they are
150 n o r m a l in the v e n t r a l h o r n s . I n c a p s a i c i n - t r e a t e d a n i m a l s , h o w e v e r , t h e r e is i n v o l v e m e n t o f the s p i n a l t r i g e m i n a l tract t h a t is s p a r e d in o u r m o d el. R e c e n t l y the effect o f n e r v e g r o w t h f a c t o r ( N G F ) o n SP at v a r i o u s levels has b e e n studied 1°.17. T h e s e results c o u l d be r e l e v a n t for the u n d e r s t a n d i n g o f the p a t h o g e n e s i s o f the disease in the m f r a t since t r e a t m e n t with N G F increases SP in g a n g l i a a n d s p i n a l d o r s a l h o r n s while it has n o s i g n i f i c a n t effect o n v e n t r a l h o r n s . The observations reported here indicate that
in the i n h e r i t e d s e n s o r y n e u r o p a t h y in the rat t h e r e is a r e d u c t i o n o f S P - i m m u n o r e a c t i v i t y in sensory pathways.
1 Barber, R. P., Vaughn, J. E., Slemmon, J. R., Salvaterra, P. M., Roberts, E. and Leeman, S. E., The origin, distribution and synaptic relationships of substance P axons in rat spinal cord,./, comp. Neurol., 184 (1979) 331-352. 2 Barbut, D., Polak, J. M. and Wall, P. D., Substance P in spinal cord dorsal horn decreases following peripheral nerve injury, Brain Research, 205 (198 !) 289-298. 3 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) 299305. 4 Chang, M. M., Leeman, S. E. and Nial, H. D., Aminoacid sequence of substance P, Nature New Biol., 232 (1971) 86-87. 5 Euler, U. S. von and Gaddum, J. H., An unidentified depressor substance in certain tissue extracts, J. Physiol. (Lond.), 72 (1931) 74-87. 6 Helke, C. J., DiMicco, J. A., Jacobowitz, D. M. and Kopin, I.J., Effects of capsaicin administration to neonatal rats on the substance P content of discrete CNS regions, Brain Research, 222 (1981) 428-431. 7 Henry, J. L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 439-451. 8 Jacobs,.J.M., Scaravilli, F., Duchen, L. W. and Mertin, J., A n~w neurological rat mutant 'mutilated foot', J. Anat., 132 (1981) 525-543. 9 JesseU, T. M., Iversen, L. L. and Cuello, A. C., Capsaicininduced depletion of substance P from primary sensory neurons, Brain Research, 152 (1978) 183-188. 10 Kessler, J. A. and Black, I. B., Nerve growth factor stimulates development of substance P in the embryonic spinal cord, Brain Research, 208 (198 !) 135 145. 11 Ljungdahl, A., H6kfelt, T. and Nilsson, G., Distribution of substance P-like immunoreactivity in the central ner-
vous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-943. 12 Murray, T. J., Congenital sensory neuropathy, Brain, 96 (1973) 387-394. 13 Nicoll, R. A., Schenker, C. and Leeman, S. E., Substance P as a transmitter candidate, Ann. Rev. Neurosci., 3 (1980) 227-268. 14 Nutt, J. G., Mroz, E. A., Leeman, S. E., Williams, A. C., Engel, W. K. and Chase, T. N., Substance P in human cerebrospinal fluid: reduction in peripheral neuropathy and autonomic dysfunction, Neurology, 30 (1980) 1280 1285. 15 Pearson, J., Familial dysautonomia (a brief review), J. A utonom. Nerv. Syst., 1 (1979) 119-126. 16 Pearson, J., Brandeis, L. and Cuello, A. C., Depletion of substance P-containing axons in substantia gelatinosa of patients with diminished pain sensitivity, Nature (Lond.), 295 (1982) 61-63. 17 Ross, M., L6fstrandh, S., Gorin, P. D., Johnson, E. M. and Schwartz, J. P., Use of an experimental autoimmune model to define nerve growth factor dependency of peripheral and central substance P-containing neurons in the rat, J. Neurosci., 1 (1981) 1304-1311. 18 Scaravilli, F. and Jacobs, J. M., Quantitative studies of motor, spinocerebellar and secondary sensory neurons in the mutilated foot mutant rat, Neuroscience, 6 (1981) 1663-1674. 19 Spillane, J. D. and Wells, C. E. C., Acrodystrophic Neuropathy, Oxford University Press, London, 1969. 20 Tessler, A., Glazer, E., Artymyshyn, R., Murray, M. and Goldberger, M. E., Recovery of substance P in the cat spinal cord after unilateral lumbosacral deafferentation, Brain Research, 191 (1980) 459-470. 21 Theiler, K., The House Mouse, Springer-Verlag, Berlin, 1972.
W e s h o u l d like to t h a n k Miss M a r g a r e t W h i t e for the h i s t o c h e m i c a l p r e p a r a t i o n s , Mr. J. A. Mills f o r h e l p with the p h o t o g r a p h y , Mrs. M a r y H a l l i d a y for the t y p i n g , a n d Dr. P. E m s o n f o r the gift o f the a n t i b o d y . This w o r k was s u p p o r t ed b y g r a n t s f r o m the N a t i o n a l F u n d f o r M e d i cal R e s e a r c h into C r i p p l i n g D i s e a s e s a n d the Medical Research Council.