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Selective degeneration by capsaicin of a subpopulation of primary sensory neurons in the adult rat
Neuroscience Letters, 59 (1985) 209-214
209
Elsevier Scientific Publishers Ireland Ltd.
NSL 03483
SELECTIVE DEGENERATION BY CAPSAICIN OF A SUBPOPULATION OF PRIMARY SENSORY NEURONS IN THE ADULT RAT
G. JANCSO j'*, ELIZABETH KIR.~LY z, F. JOO 3, G. SUCH I and ANIKO NAGY I
Departments of JPhysiology and 2Anatomy, University Medical School, D/Jm t~r 10.. H-6720 Szeged," and 3Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged (Hungary) (Received March 25th, 1985; Revised version received May 20th, 1985; Accepted May 29th, 1985)
Key words: capsaicin - degeneration - primary sensory neuron - adult rat
The morphological effects of systemic capsaicin treatment have been studied in adult rats. Light and electron microscopy revealed that a subpopulation of small-to-medium sized B-type primary sensory neurons, representing about 17% of the total neuronal population in the 4th lumbar spinal ganglion, underwent rapid degeneration after the administration of capsaicin. Quantitative electron microscopy demonstrated a decrease of about 45~ in the number of unmyelinated axons in the saphenous nerve. Light microscopy showed extensive axon terminal degeneration in the brainstem and spinal cord confined to the central projection areas of capsaicin-sensitive afferent fibers, as has already been revealed in the newborn rat. The present results furnish evidence for a hitherto unrecognized selective neurodegenerative action of capsaicin in the adult rat.
In recent years capsaicin became a widely used specific pharmacological tool in neurobiological research devoted to the better understanding of different facets of the morphological and functional organization of the sensory nervous system [5, 12, 18, 22]. Capsaicin given to newborn rats results in an irreversible loss of specific sensory functions [2, 6, 12, 18, 20, 23, 24] by inducing a selective degeneration of about 50% of sensory ganglion cells [17, 22] which correspond to B-type [1], peptide-conraining, primary afferent neurons [6, 10, 12, 20]. In adult rats, however, previous morphological studies revealed only minor ultrastructural changes in certain sensory ganglion cells after capsaicin treatment [15]. Considering the selective neurodegenerarive action of capsaicin in newborn animals [12], the aim of the present experiments was to examine the possibility whether some of the apparently irreversible effects of capsaicin in adult rats [13] may result from degeneration of primary sensory neurons. Adult male rats of the Sprague-Dawley CFY strain weighing 200-250 g were used. One group of animals was injected subcutaneously (s.c.) with capsaicin at a dose of 100 mg/kg and killed 1-6 h after treatment. Rats were perfused with a fixative containing 2% paraformaldehyde-l% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, and the gasserian ganglia, lumbar spinal ganglia and lumbar dorsal roots dissected *Author for correspondence. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
210 OUt a n d p r o c e s s e d for e l e c t r o n m i c r o s c o p i c e x a m i n a t i o n . A n i m a l s r e c e i v i n g a s i m i l a r a m o u n t o f the s o l v e n t for c a p s a i c i n (10°/,, e t h a n o l - 1 0 ° / T w e e n
80 in p h y s i o l o g i c a l
saline) s e r v e d as c o n t r o l s . A n o t h e r g r o u p o f rats r e c e i v e d a s.c. i n j e c t i o n o f 100 m g / k g o f c a p s a i c i n a n d w e r e p e r f u s e d w i t h the a b o v e f i x a t i v e 120 m i n later. T h e 4th l u m b a r spinal g a n g l i a w e r e d i s s e c t e d o u t , s t o r e d in the f i x a t i v e for 12 h, w a s h e d in buffer, o s m i c a t e d w i t h a 2~ o b u f f e r e d s o l u t i o n o f OsO4 for 4 h, at 4'~C, d e h y d r a t e d in a l c o h o l s a n d finally e m b e d d e d in A r a l d i t e . F o r the a s s e s s m e n t o f n e u r o n a l cell n u m b e r s in t h a t g a n g l i a , serial s e c t i o n s (3 u m in t h i c k n e s s ) w e r e c u t o n a P y r a m i t o m e ( L K B ) a n d s t a i n e d w i t h a s o l u t i o n c o n t a i n i n g 0 . 1 ~ m e t h y l e n e blue-0.1~,~, t h i o n i n e . T h e use o f
Fig. I. Structural changes produced by a single injection of capsaicin (100 mg/kg, s.c.) to adult rats. a: light microscopic photograph taken from the 4th lumbar spinal ganglion 2 h after treatment. A small-sized neuron exhibits an irregular, pyknotic nucleus, increased nuclear and cytoplasmic basophilia and severe cytoplasmic vacuolization. The structure of a large-sized neuron is apparently intact, x 559. b: electron micrograph showing a detail of a B-type sensory ganglion cell 2 h after the injection of capsaicin. The fine structure of this neuron is severly impaired; the nucleus exhibits irregular indentations and dissolution of the chromatin substance, the cellular organelles could hardly be identified within the cytoplasm having an enhanced electron density, x 4680. c: presence of argyrophilic particles indicating heavy axon terminal degeneration in the superficial layers (Rexed's laminae I and ll) of the dorsal horn of the lumbar spinal cord 8 h after the administration of capsaicin. Roman numerals indicate Rexed's laminae I--I11. Eager's method, x 325. d: electron micrograph showing degenerated unmyelinated axons in the 4th lumbar spinal dorsal root 4 h after capsaicin treatment. × 16.380
211
3 am thick sections was necessary to assess the precise cytological characteristics of the damaged neurons. Ganglion cells exhibiting a clearly identifiable nucleolus were counted according to the ocular square method at a magnification of x 400. Criteria for degenerating neurons were: the presence of a pyknotic, irregularly outlined nucleus; increased nuclear and cytoplasmic basophilia; and severe vacuolization in the perikaryon. Normal and degenerating neurons were counted separately in every 10th section. The number of nucleoli were converted to ganglion cell number according to formula 4 of K6nigsmark [16]. Multiplying this value by 10 yielded the actual number of neurons in the ganglion. In another series of experiments rats were given either a single s.c. injection of 50 or 100 mg/kg of capsaicin or injected with a single s.c. dose of 100 mg/kg ofcapsaicin followed by a second injection of a 200 mg/kg dose of the drug 6 days later. Animals were killed 30 days after the last injection and specimens of the saphenous nerves were processed for electron microscopic examination. Counts of all axons were made on montages of the entire cross-section of the nerves composed of electron micrographs taken at an original magnification of x 2000 and enlarged photographically to x 5400. For the demonstration of axon terminal degeneration, animals were perfused with 109/o formalin 7-12 h after capsaicin (100 mg/kg) treatment. Frozen sections were cut from the brainstem and the spinal cord and processed according to the method of Eager [4]. Administration of capsaicin at a dose of 100 mg/kg induced severe structural alterations in certain neurons of the trigeminal and spinal sensory ganglia which could easily be detected even by light microscopic examination. Numerous small-tomedium sized sensory ganglion cells exhibited an increased nuclear and cytoplasmic basophilia, inhomogeneous staining of the shrunken, pyknotic nucleus, and marked cytoplasmic vacuolization (Fig. la). Electron microscopy confirmed and extended these findings. In sensory ganglia of rats killed !-2 h after the administration of capsaicin, the most prominent features of the damaged neurons, belonging to B-type sensory ganglion cells according to morphological criteria [I], were the enhanced electron density of both the nucleus and the perikaryon, mitochondrial swelling and the dilatation of the cisternae of the rough endoplasmic reticulum and of the Golgi apparatus. Degenerative changes advanced in time; after a survival of 3-4 h cellular organelles could hardly be identified, and the nuclear membrane was often disrupted TABLE i T H E E F F E C T OF C A P S A I C I N (100 mg/kg) ON N E U R O N A L C E L L N U M B E R S IN T H E L E F T 4th L U M B A R SPINAL G A N G L I O N O F 3 A D U L T RATS N u m b e r of dorsal root ganglion neurons Total
Degenerating
6674 6783 6634
1145 1185 1165
Percent o f degenerating neurons
17.1 17.4 17.5
212
(Fig. lb). These findings indicate that administration of capsaicin in the adult rat induces progressive rapid deterioration of the structural integrity of some B-type primary sensory neurons, leading to the degeneration of these cells. In control animals, injection of the solvent for capsaicin did not induce any change in the cellular morphology of sensory ganglia. Quantitative evaluation of the number of damaged ganglion cells revealed that about 17~o of the total neuronal population underwent degeneration in the 4th lumbar spinal ganglion after an injection of capsaicin at a dose of 100 mg/kg (Table I). By using a selective silver impregnation technique, heavy axon terminal degeneration was demonstrated in the superficial layers of the trigeminal nucleus caudalis and the spinal cord dorsal horn, as well as in the nucleus of the solitary tract, 7--I 2 h after capsaicin treatment (Fig. lc). It is reasonable to assume that degenerating axonal endings represent the central terminations of the affected sensory neurons because many, mainly unmyelinated, degenerating axons were seen in dorsal roots 4-6 h after capsaicin treatment (Fig. ld). In addition, the distribution of degenerating axon terminals (to be reported elsewhere in detail) corresponds closely to the projection areas of capsaicin-sensitive primary afferents, as revealed in the newborn rat
[ll]. In order to further assess the extent of the degeneration produced by capsaicin in the adult rat, the number of axons was counted in the saphenous nerves of control and capsaicin-treated animals. Quantitative electron microscopy disclosed that capsaicin treatment resulted in a dose-dependent substantial decrease in the numbers of TABLE II AXONAL COUNTS OF THE SAPHENOUS NERVES OF A D U L T RATS TREATED WITH DIFFERENT DOSES OF CAPSAICIN In each group values represent the number of axons counted in the saphenous nerves of 3 rats. Number of axons Myelinated
Unmyelinated
Control
885 897 878
4260 4390 4263
Capsaicin 50 mg/kg
793 858 833
2823 3118 3139
Capsaicin 100 mg/kg
727 773 733
2514 2259 2743
Capsaicin 100+200 mg/kg
758 779 739
2107 2182 2370
213 both myelinated and unmyelinated fibers (Table II). However, a further increase of the dose o f capsaicin (100 and 100 + 200 mg/kg, respectively, to the same animal) did not result in a significant difference in the reduction o f the number o f myelinated and unmyelinated axons. Accordingly, these findings indicate that in the adult rat, the neurodegenerative action of capsaicin is restricted only to a subpopulation of primary sensory neurons, since even a 3-fold increase of the dose o f capsaicin failed to cause a significantly higher incidence o f degenerating sensory ganglion cells which possess unmyelinated axons. It is to be noted in this respect that in the newborn rat, administration of capsaicin at a dose of 50 mg/kg results in an about 50 and 70~o reduction in the numbers o f dorsal root ganglion neurons and unmyelinated axons of the saphenous nerve, respectively [ 12, 17, 2 !]. Although some previous experimental data already pointed out the possibility that capsaicin may cause destruction of sensory terminals in adult rats [3, 9, 10], degeneration of the entire sensory neuron has not been demonstrated. Hence, it is generally believed that in adults the effects of capsaicin may be connected with an impairment of axon transport mechanisms in, and a depletion o f neuropeptides from, primary afferent neurons [7, ! 4, ! 8, 19, 21]. In light of the present results, it seems likely that some of the long-lasting, apparently irreversible effects o f capsaicin may be attributed to degeneration, while others may result from the depletion of neuropeptides. It is worthy of note in this respect that preliminary experiments show a similar degeneration of sensory ganglion cells in species other than rodents (Jancs6, Such and Nagy, unpublished observations). In conclusion, the results presented here indicate the existence o f a special subpopulation of capsaicin-sensitive primary sensory neurons in the adult rat which may be distinguished by their selective vulnerability to the neurodegenerative action of capsaicin. The findings may point to new possibilities in the study of the morphological and functional organization as well as the neuroehemical characterization of primary sensory neurons, and necessitate a re-evaluation of previous experimental results obtained with capsaicin in adult animals. This work was supported in part by a grant from the Scientific Research Council, Hungarian Ministry of Health (Grant 4-05-0303-03/2) and by TPB 48. We thank H. Szab6 and K. Mohficsi for technical assistance. I Andres, K.H., Untersuchungenfiberden Feinbau von Spinalganglien,Z. Zellforsch.,55 (1961) 1--48. 2 Cervero, F. and McRitchie, H.A., Neonatal capsaicin and thermal nociception:a paradox, Brain Res., 215 (1981)414-418. 3 Chung, K., Sehwen, R.J. and Coggeshall, R.E., Ureteral axon damage following subcutaneous administration of capsaicin in adult rats, Neurosci. Len., 53 (1985) 221-226. 4 Eager, R.P., Selectivestaining of degeneratingaxons in the central nervoussystemby a simplifiedsilver method: spinal cord projections to external cuneate and inferior olivary nuclei in the cat, Brain Res., 22 (1970) 137-141. 5 Fitzgerald,M., Capsaicin and sensoryneurons. A review,Pain, 15 (1983) 109-130. 6 Gamse, R., Holzer, P. and Lembeck,F., Decrease of substance P in primary afferent neurones and impairment of neurogenicplasma extravasationby capsaicin, Br. J. Pharmacol.,68 (I 980) 207-213.
214 7 Gamse, R., Lackner, D., Gamse, G. and Leeman, S.E., Effect of capsaicin pretreatment on capsaicinevoked release of immunoreactive somatostatin and substance P from primary sensory neurons. Naunyn-Schmiedeberg's Arch. Pharmacol., 316 (1981) 19-24. 8 Hoyes, A.D. and Barber, P., Degeneration of axons in the ureteric and duodenal nerve plexuses of the adult rat following in vivo treatment with capsaicin, Neurosci. Lett., 24 0981) 19- 24. 9 Hoyes, A.D., Barber, P. and Jagessar, H., Effect of capsaicin on the intraperitoneal axons of the rat trachea, Neurosci. Lett., 26 ( 198 I) 329-334. l0 Jancs6, G., Hfkfelt, T., Lundberg, J.M., Kir',ily, E., Halfi.sz, N., Nilsson, G., Terenius, L., Rehfeld, J., Steinbusch, 1t., Verhofstad, A., Elde, R., Said, S. and Brown, M., Immunohistochemical studies on the effect of capsaicin on spinal and medullary peptide and monoamine neurons using antisera to substance P. gastrin/CCK, somatostatin, VIP, enkephalin, neurotensin and 5-hydroxytryptamine, J. Neurocytol., 10 ( 19811 963-980. 11 Jancs6, G. and Kir',ily, E.. Distribution of chemosensitive primary sensory afferents in the central nervous system of the rat, J. Comp. Neurol., 190 (1980) 781-792. 12 Jancs6, G., Kir~ly, E. and Jancs6-G~bor, A., Pharmacologically induced selective degeneration ofchemosensitive primary sensory neurones, Nature (Lond.), 270 (1977) 741-743. 13 Jancs6-G:~bor, A., Szolc~nyi, J. and Jancso, N., Irreversible impairment of thermoregulation induced by capsaicin and similar pungent substances, J. Physiol. (Lond.), 206 (1970) 495--507. 14 Jessel, T.M., Iversen, L.L. and Cuello, A.C., Capsaicin-induced depletion of substance P from primary sensory neurones, Brain Res., 152 (1978) 183-188. 15 Joo, F., Szolcs~inyi, J. and Jancs6-G~ibor, A., Mitochondrial alterations in the spinal ganglion cells of the rat accompanying the long-lasting ,sensory disturbance induced by capsaicin, Life Sci., 8 (1969) 621- 626. 16 K6nigsmark, B.W., Methods for the counting of neurons. In W.J.H. Nauta and S.O.E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 315-340. 17 I,awson, S.N. and Nickels, S.M., The use of morphometric techniques to analyze the effect of neonatal capsaicin treatment on rat dorsal root ganglia and dorsal roots, J. Physiol. (Lond.), 303 (1980) 12P. 18 Lembeck, F. and Gamse, R., Substance P in peripheral sensory processes. In R. Porter and M. O'Connor (Eds.), Substance P in the Nervous System, Pitman, London, 1982, pp. 35-54. 19 Miller, M.S., Buck, S.H., Sipes, I.G., Yamamura, H.I. and Burks T.F., Regulation of substance P by nerve growth factor: disruption by capsaicin, Brain Res., 250 (1982) 193-196. 20 Nagy, J.l., Vincent, S.R., Staines, W.A., Fibiger, H.C., Reisine, T.D. and Yamamura, H.L., Neurotoxic action of capsaicin on spinal substance P neurons, Brain Res., 186 (1980) 435-444. 21 Otten. U., Lorenz, H.P. and Businger, F., Nerve growth factor antagonizes the neurotoxic action of capsaicin on primary sensory neurones, Nature (Lond.), 301 (1983) 515-517. 22 Szolc~nyi, J., Capsaicin type pungent agents producing pyrexia. In A.S. Milton (Ed.), Handbook of Experimental Pharmacology, Vol. 60, Springer, Berlin, 1982, pp. 437-478. 23 Wall, P.D., Fitzgerald, M., Nussbaumer, J.C., Van der Loos, M. and Devor, M., Somatotopic maps are disorganized in adult rodents treated with capsaicin as neonates, Nature (Lond.), 295 (1982) 691693. 24 Welk, E., Fleischer, F., Petsche, U. and Handwerker, H.O., Afferent C-fibres in rats after neonatal capsaicin treatment, Naunyn-Schmiedeberg's Arch. Pharmacol., 400 (1984) 66-71.
209
Elsevier Scientific Publishers Ireland Ltd.
NSL 03483
SELECTIVE DEGENERATION BY CAPSAICIN OF A SUBPOPULATION OF PRIMARY SENSORY NEURONS IN THE ADULT RAT
G. JANCSO j'*, ELIZABETH KIR.~LY z, F. JOO 3, G. SUCH I and ANIKO NAGY I
Departments of JPhysiology and 2Anatomy, University Medical School, D/Jm t~r 10.. H-6720 Szeged," and 3Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged (Hungary) (Received March 25th, 1985; Revised version received May 20th, 1985; Accepted May 29th, 1985)
Key words: capsaicin - degeneration - primary sensory neuron - adult rat
The morphological effects of systemic capsaicin treatment have been studied in adult rats. Light and electron microscopy revealed that a subpopulation of small-to-medium sized B-type primary sensory neurons, representing about 17% of the total neuronal population in the 4th lumbar spinal ganglion, underwent rapid degeneration after the administration of capsaicin. Quantitative electron microscopy demonstrated a decrease of about 45~ in the number of unmyelinated axons in the saphenous nerve. Light microscopy showed extensive axon terminal degeneration in the brainstem and spinal cord confined to the central projection areas of capsaicin-sensitive afferent fibers, as has already been revealed in the newborn rat. The present results furnish evidence for a hitherto unrecognized selective neurodegenerative action of capsaicin in the adult rat.
In recent years capsaicin became a widely used specific pharmacological tool in neurobiological research devoted to the better understanding of different facets of the morphological and functional organization of the sensory nervous system [5, 12, 18, 22]. Capsaicin given to newborn rats results in an irreversible loss of specific sensory functions [2, 6, 12, 18, 20, 23, 24] by inducing a selective degeneration of about 50% of sensory ganglion cells [17, 22] which correspond to B-type [1], peptide-conraining, primary afferent neurons [6, 10, 12, 20]. In adult rats, however, previous morphological studies revealed only minor ultrastructural changes in certain sensory ganglion cells after capsaicin treatment [15]. Considering the selective neurodegenerarive action of capsaicin in newborn animals [12], the aim of the present experiments was to examine the possibility whether some of the apparently irreversible effects of capsaicin in adult rats [13] may result from degeneration of primary sensory neurons. Adult male rats of the Sprague-Dawley CFY strain weighing 200-250 g were used. One group of animals was injected subcutaneously (s.c.) with capsaicin at a dose of 100 mg/kg and killed 1-6 h after treatment. Rats were perfused with a fixative containing 2% paraformaldehyde-l% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, and the gasserian ganglia, lumbar spinal ganglia and lumbar dorsal roots dissected *Author for correspondence. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
210 OUt a n d p r o c e s s e d for e l e c t r o n m i c r o s c o p i c e x a m i n a t i o n . A n i m a l s r e c e i v i n g a s i m i l a r a m o u n t o f the s o l v e n t for c a p s a i c i n (10°/,, e t h a n o l - 1 0 ° / T w e e n
80 in p h y s i o l o g i c a l
saline) s e r v e d as c o n t r o l s . A n o t h e r g r o u p o f rats r e c e i v e d a s.c. i n j e c t i o n o f 100 m g / k g o f c a p s a i c i n a n d w e r e p e r f u s e d w i t h the a b o v e f i x a t i v e 120 m i n later. T h e 4th l u m b a r spinal g a n g l i a w e r e d i s s e c t e d o u t , s t o r e d in the f i x a t i v e for 12 h, w a s h e d in buffer, o s m i c a t e d w i t h a 2~ o b u f f e r e d s o l u t i o n o f OsO4 for 4 h, at 4'~C, d e h y d r a t e d in a l c o h o l s a n d finally e m b e d d e d in A r a l d i t e . F o r the a s s e s s m e n t o f n e u r o n a l cell n u m b e r s in t h a t g a n g l i a , serial s e c t i o n s (3 u m in t h i c k n e s s ) w e r e c u t o n a P y r a m i t o m e ( L K B ) a n d s t a i n e d w i t h a s o l u t i o n c o n t a i n i n g 0 . 1 ~ m e t h y l e n e blue-0.1~,~, t h i o n i n e . T h e use o f
Fig. I. Structural changes produced by a single injection of capsaicin (100 mg/kg, s.c.) to adult rats. a: light microscopic photograph taken from the 4th lumbar spinal ganglion 2 h after treatment. A small-sized neuron exhibits an irregular, pyknotic nucleus, increased nuclear and cytoplasmic basophilia and severe cytoplasmic vacuolization. The structure of a large-sized neuron is apparently intact, x 559. b: electron micrograph showing a detail of a B-type sensory ganglion cell 2 h after the injection of capsaicin. The fine structure of this neuron is severly impaired; the nucleus exhibits irregular indentations and dissolution of the chromatin substance, the cellular organelles could hardly be identified within the cytoplasm having an enhanced electron density, x 4680. c: presence of argyrophilic particles indicating heavy axon terminal degeneration in the superficial layers (Rexed's laminae I and ll) of the dorsal horn of the lumbar spinal cord 8 h after the administration of capsaicin. Roman numerals indicate Rexed's laminae I--I11. Eager's method, x 325. d: electron micrograph showing degenerated unmyelinated axons in the 4th lumbar spinal dorsal root 4 h after capsaicin treatment. × 16.380
211
3 am thick sections was necessary to assess the precise cytological characteristics of the damaged neurons. Ganglion cells exhibiting a clearly identifiable nucleolus were counted according to the ocular square method at a magnification of x 400. Criteria for degenerating neurons were: the presence of a pyknotic, irregularly outlined nucleus; increased nuclear and cytoplasmic basophilia; and severe vacuolization in the perikaryon. Normal and degenerating neurons were counted separately in every 10th section. The number of nucleoli were converted to ganglion cell number according to formula 4 of K6nigsmark [16]. Multiplying this value by 10 yielded the actual number of neurons in the ganglion. In another series of experiments rats were given either a single s.c. injection of 50 or 100 mg/kg of capsaicin or injected with a single s.c. dose of 100 mg/kg ofcapsaicin followed by a second injection of a 200 mg/kg dose of the drug 6 days later. Animals were killed 30 days after the last injection and specimens of the saphenous nerves were processed for electron microscopic examination. Counts of all axons were made on montages of the entire cross-section of the nerves composed of electron micrographs taken at an original magnification of x 2000 and enlarged photographically to x 5400. For the demonstration of axon terminal degeneration, animals were perfused with 109/o formalin 7-12 h after capsaicin (100 mg/kg) treatment. Frozen sections were cut from the brainstem and the spinal cord and processed according to the method of Eager [4]. Administration of capsaicin at a dose of 100 mg/kg induced severe structural alterations in certain neurons of the trigeminal and spinal sensory ganglia which could easily be detected even by light microscopic examination. Numerous small-tomedium sized sensory ganglion cells exhibited an increased nuclear and cytoplasmic basophilia, inhomogeneous staining of the shrunken, pyknotic nucleus, and marked cytoplasmic vacuolization (Fig. la). Electron microscopy confirmed and extended these findings. In sensory ganglia of rats killed !-2 h after the administration of capsaicin, the most prominent features of the damaged neurons, belonging to B-type sensory ganglion cells according to morphological criteria [I], were the enhanced electron density of both the nucleus and the perikaryon, mitochondrial swelling and the dilatation of the cisternae of the rough endoplasmic reticulum and of the Golgi apparatus. Degenerative changes advanced in time; after a survival of 3-4 h cellular organelles could hardly be identified, and the nuclear membrane was often disrupted TABLE i T H E E F F E C T OF C A P S A I C I N (100 mg/kg) ON N E U R O N A L C E L L N U M B E R S IN T H E L E F T 4th L U M B A R SPINAL G A N G L I O N O F 3 A D U L T RATS N u m b e r of dorsal root ganglion neurons Total
Degenerating
6674 6783 6634
1145 1185 1165
Percent o f degenerating neurons
17.1 17.4 17.5
212
(Fig. lb). These findings indicate that administration of capsaicin in the adult rat induces progressive rapid deterioration of the structural integrity of some B-type primary sensory neurons, leading to the degeneration of these cells. In control animals, injection of the solvent for capsaicin did not induce any change in the cellular morphology of sensory ganglia. Quantitative evaluation of the number of damaged ganglion cells revealed that about 17~o of the total neuronal population underwent degeneration in the 4th lumbar spinal ganglion after an injection of capsaicin at a dose of 100 mg/kg (Table I). By using a selective silver impregnation technique, heavy axon terminal degeneration was demonstrated in the superficial layers of the trigeminal nucleus caudalis and the spinal cord dorsal horn, as well as in the nucleus of the solitary tract, 7--I 2 h after capsaicin treatment (Fig. lc). It is reasonable to assume that degenerating axonal endings represent the central terminations of the affected sensory neurons because many, mainly unmyelinated, degenerating axons were seen in dorsal roots 4-6 h after capsaicin treatment (Fig. ld). In addition, the distribution of degenerating axon terminals (to be reported elsewhere in detail) corresponds closely to the projection areas of capsaicin-sensitive primary afferents, as revealed in the newborn rat
[ll]. In order to further assess the extent of the degeneration produced by capsaicin in the adult rat, the number of axons was counted in the saphenous nerves of control and capsaicin-treated animals. Quantitative electron microscopy disclosed that capsaicin treatment resulted in a dose-dependent substantial decrease in the numbers of TABLE II AXONAL COUNTS OF THE SAPHENOUS NERVES OF A D U L T RATS TREATED WITH DIFFERENT DOSES OF CAPSAICIN In each group values represent the number of axons counted in the saphenous nerves of 3 rats. Number of axons Myelinated
Unmyelinated
Control
885 897 878
4260 4390 4263
Capsaicin 50 mg/kg
793 858 833
2823 3118 3139
Capsaicin 100 mg/kg
727 773 733
2514 2259 2743
Capsaicin 100+200 mg/kg
758 779 739
2107 2182 2370
213 both myelinated and unmyelinated fibers (Table II). However, a further increase of the dose o f capsaicin (100 and 100 + 200 mg/kg, respectively, to the same animal) did not result in a significant difference in the reduction o f the number o f myelinated and unmyelinated axons. Accordingly, these findings indicate that in the adult rat, the neurodegenerative action of capsaicin is restricted only to a subpopulation of primary sensory neurons, since even a 3-fold increase of the dose o f capsaicin failed to cause a significantly higher incidence o f degenerating sensory ganglion cells which possess unmyelinated axons. It is to be noted in this respect that in the newborn rat, administration of capsaicin at a dose of 50 mg/kg results in an about 50 and 70~o reduction in the numbers o f dorsal root ganglion neurons and unmyelinated axons of the saphenous nerve, respectively [ 12, 17, 2 !]. Although some previous experimental data already pointed out the possibility that capsaicin may cause destruction of sensory terminals in adult rats [3, 9, 10], degeneration of the entire sensory neuron has not been demonstrated. Hence, it is generally believed that in adults the effects of capsaicin may be connected with an impairment of axon transport mechanisms in, and a depletion o f neuropeptides from, primary afferent neurons [7, ! 4, ! 8, 19, 21]. In light of the present results, it seems likely that some of the long-lasting, apparently irreversible effects o f capsaicin may be attributed to degeneration, while others may result from the depletion of neuropeptides. It is worthy of note in this respect that preliminary experiments show a similar degeneration of sensory ganglion cells in species other than rodents (Jancs6, Such and Nagy, unpublished observations). In conclusion, the results presented here indicate the existence o f a special subpopulation of capsaicin-sensitive primary sensory neurons in the adult rat which may be distinguished by their selective vulnerability to the neurodegenerative action of capsaicin. The findings may point to new possibilities in the study of the morphological and functional organization as well as the neuroehemical characterization of primary sensory neurons, and necessitate a re-evaluation of previous experimental results obtained with capsaicin in adult animals. This work was supported in part by a grant from the Scientific Research Council, Hungarian Ministry of Health (Grant 4-05-0303-03/2) and by TPB 48. We thank H. Szab6 and K. Mohficsi for technical assistance. I Andres, K.H., Untersuchungenfiberden Feinbau von Spinalganglien,Z. Zellforsch.,55 (1961) 1--48. 2 Cervero, F. and McRitchie, H.A., Neonatal capsaicin and thermal nociception:a paradox, Brain Res., 215 (1981)414-418. 3 Chung, K., Sehwen, R.J. and Coggeshall, R.E., Ureteral axon damage following subcutaneous administration of capsaicin in adult rats, Neurosci. Len., 53 (1985) 221-226. 4 Eager, R.P., Selectivestaining of degeneratingaxons in the central nervoussystemby a simplifiedsilver method: spinal cord projections to external cuneate and inferior olivary nuclei in the cat, Brain Res., 22 (1970) 137-141. 5 Fitzgerald,M., Capsaicin and sensoryneurons. A review,Pain, 15 (1983) 109-130. 6 Gamse, R., Holzer, P. and Lembeck,F., Decrease of substance P in primary afferent neurones and impairment of neurogenicplasma extravasationby capsaicin, Br. J. Pharmacol.,68 (I 980) 207-213.
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