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Increased staining of immunoreactive dynorphin cell bodies in the deafferented spinal cord of the rat
Neuroscience Letters, 84 (I 988) 125~ 130 Elsevier Scientific Publishers Ireland Ltd.
125
NSL 05056
Increased staining of immunoreactive dynorphin cell bodies in the deafferented spinal cord of the rat Hee Jung Cho and Allan I. Basbaum Departments o[Anatomy and Physiology. University of Cali]brnia San Francisco. San Francisco. ('A 94143 (U.S.A.)
(Received 14 August 1987: Revised version received 22 September 1987; Accepted 29 September 1987} Key wor¢Lw Dynorphin: lmmunocytochemistry; Deafferentation: Dorsal horn; Pain: Rat
This study examined the distribution of immunoreactive dynorphin neurons in the lumbar dorsal horn of unilaterally deafferented, colchicine-treated rats. Ipsilateral to a multiple dorsal rhizolomy there was a significant increase both in the number and intensity of staining of dynorphin-immunoreactive cells in laminae I, outer II and V. A comparable change was seen in animals that were deafferented by sciatic nerve section. Enkephalin immunoreactivity was not altered under these conditions. These results indicate that many forms of injury, not all of which result in increased nociceptive input, can increase lhe level ofdynorphin in spinal cord neurons.
I m m u n o c y t o c h e m i c a l studies have identified significant differences in the spinal c o r d d i s t r i b u t i o n o f the two o p i o i d peptides, e n k e p h a l i n a n d d y n o r p h i n , M o s t n o t a bly, there is a relatively restricted d i s t r i b u t i o n o f d y n o r p h i n cell bodies, in l a m i n a e I, in the o u t e r p a r t o f the s u b s t a n t i a g e l a t i n o s a ( l a m i n a IIo) a n d in l a m i n a V [7, 15, 19, 24]. E n k e p h a l i n cell b o d i e s are m o r e widely d i s t r i b u t e d . T h e y are f o u n d in all layers o f the superficial d o r s a l horn, including the inner p a r t o f the s u b s t a n t i a gelatinosa and in l a m i n a lII, two regions typically a s s o c i a t e d with the processing o f inform a t i o n related to n o n - n o x i o u s inputs [1]. T h e r e is a c o r r e s p o n d i n g difference in the t e r m i n a l d i s t r i b u t i o n o f the two peptides. D y n o r p h i n t e r m i n a l s are c o n c e n t r a t e d in l a m i n a e l a n d V: e n k e p h a l i n t e r m i n a l s are m o r e widely d i s t r i b u t e d . Finally, some p r i m a r y afferents ( p a r t i c u l a r l y in the sacral spinal c o r d o f the cat) c o n t a i n d y n o r p h i n [2, 4, 23]. D o r s a l r h i z o t o m y decreases the t e r m i n a l d y n o r p h i n i m m u n o r e a c t i v i t y in the sacral spinal cord; as expected, there are m i n i m a l c h a n g e s in e n k e p h a l i n levels
[21 W i t h the exception o f a small b u l b o s p i n a l e n k e p h a l i n p r o j e c t i o n [5, 10, 17], the m a j o r i t y o f spinal e n k e p h a l i n arises f r o m spinal i n t e r n e u r o n s . T h e latter are h y p o t h e sized to exert a segmental a n t i n o c i c e p t i v e c o n t r o l via b o t h pre- a n d p o s t s y n a p t i c inhiCorrespmTdence: A.I. Basbaum, Departments of Anatomy and Physiology, University of California San
Francisco, San Francisco, CA 94143, U.S.A.
126
bition of spinal nociceptive neurons [14]. Anatomical studies, however, indicate that axodendritic enkephalin relationships predominate [9, 20]. To date, the relationship ofdynorphin terminals to primary afferent fibers has not been addressed. With a view to characterizing the synaptic relationship between degenerating primary afferent fibers and the intrinsic dynorphin cell population of the superficial dorsal horn, we have repeated the dorsal rhizotomy studies in rats that were treated with colchicine so that the local dynorphin cells of the dorsal horn could be visualized. This report describes a striking increase in dynorphin perikaryal immunoreactivity in the colchicine-treated, deafferented dorsal horn. Male rats were anesthetized with Nembutal (60 mg/kg) and a laminectomy was performed over the lumbosacral enlargement. At the caudal end of the enlargement the dura was incised and reflected laterally so as to expose the incoming dorsal roots. Dorsal roots Ls-S1 were then cut with iridectomy scissors. To increase the staining of dynorphin cell bodies, these studies were performed in colchicine-treated rats. After the roots were cut, a 10-/zl Hamilton syringe with a fixed 34-gauge needle was threaded under the dura, rostral to the rhizotomy, until the tip of the needle was located over the lumbar enlargement. Ten/A of a 20/zg/~tl solution of colchicine was injected slowly and then the needle was withdrawn. At no time did the needle come into contact with the cord surface. When the injection was completed, the animal was tilted head down to minimize caudal diffusion of the colchicine. After 3 min, a strip of moistened Gelfilm was placed over the exposed cord, the overlying muscle and skin were sutured and the rats returned to their cages. Twenty-four hours later the rats were reanesthetized and perfused intracardially with the following solutions: 100 ml of a 0.1 M phosphate-buffered saline wash and 400 ml of a fixative containing 0.1 M phosphate-buffered 4% paraformaldehyde, and in some cases 0.1% glutaraldehyde. The appropriate spinal cord segments were removed and the completeness of the rhizotomy verified under a dissecting microscope. After washing the tissue in phosphate-buffer (PB), fifty micron Vibratome sections through the lumbar enlargement were cut and collected into PB. The sections were then processed with the avitin-biotin method for localizing immunoreactive dynorphin. An antiserum directed against dynorphin B (a kind gift of Dr. Eckard Weber) was used at a dilution of i:3000. Some sections were osmicated and embedded in plastic for subsequent electron microscopic analysis. A detailed description of the synaptic relationship between immunoreactive dynorphin B cell bodies and terminals and degenerating primary afferent terminals will be described in another report. In two additional rats, the sciatic nerve was exposed at the sciatic notch and a ligature tied around it. At least 1 cm of the distal stump was cut and removed. One of the rats was administered colchicine intrathecally at the time of the peripheral denervation and was perfused 24 h later. The other rat was administered colchicine 6 days after the sciatic nerve section and was perfused 24 h later. It is rare to find immunoreactive dynorphin perikarya in the spinal cord of the normal rat. Twenty-four hours after colchicine treatment, however, there were large numbers of dynorphin cells labelled bilaterally in the lumbosacral enlargement. The
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distribution of cells corresponded to those segments which were topically exposed to the colchicine. The majority of labelled cells were found in the superficial layers of the dorsal horn, laminae I and outer II. Fewer, but larger cells were found in the region of lamina V. Approximately equal numbers of labelled neurons were found on both sides of the cord; there was no detectable difference in the intensity of staining on the two sides. In rats that had undergone a multiple dorsal rhizotomy at the time of the colchicine administration, there was considerable asymmetry in the staining of the dynorphin cell bodies. Ipsilateral to the deafferentation there was a large increase in both the number and intensity of immunoreactive dynorphin cell bodies. The distribution of labelled cells, however, did not differ on the two sides of the cord. The increased perikaryal staining was found in the same region where labelled dynorphin cells are found in the unoperated animal, i.e. in the superficial dorsal horn and in the region of lamina V. Fig. 1 is taken from an osmicated, plastic embedded Vibratome section and illustrates that increased perikaryal staining can be seen across the mediolateral extent of the superficial dorsal horn, particularly in laminae I and outer II.
Fig. 1. lmmunoreactive dynorphin cells in the lumbar spinal cord of a unilaterally deafferented, colchicinetreated rat. The increased cell staining is evident in the superficial dorsal horn (laminae I and outer I1) of the deafferented (right) side of the cord (A). The areas outlined in the left and right boxes are magnified in B and C, respectively. Note that there is not only an increased number of immunoreactive dynorphin cells labelled on the deafferented side of the cord, but the density of the staining is also increased. Arrowheads in B and C identify immunoreactive dynorphin cell bodies. I, 11o and Ili identify lamina I, and the outer and inner portions of the substantia gelatinosa, respectively. Bars = 2 0 0 / l m in A and 50 t~m in B and C.
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To provide a quantitative estimate of the increased staining on the deafferented side, counts of labelled neurons were made in 15 sections through the lumbar enlargement of one rat. The sections from this rat were immunoreacted without Triton in the incubation media and were osmicated and embedded in plastic. This limited the terminal staining and made it much simpler to identify and count labelled cells. Cells were counted in two focal planes, corresponding to the two surfaces of the section. The average number of immunoreactive dynorphin cells in lamina I on the unoperated side was 24.6. On the deafferented side there was an average of 34.1 labelled cells. In lamina V, there was an average of 2.2 labelled cells on the unoperated side and 5.1 cells on the deafferented side. We also counted cells from unosmicated sections through the lumbar cord of the rats that underwent sciatic nerve section. Increased perikaryal staining was found in the dorsal horn ipsilateral to a sciatic nerve section. In the rat that survived 24 h after sciatic nerve section, there was an average of 13.8 labelled cells in laminae I and I1 per 50/zm Vibratome section on the unoperated side and 20.1 on the denervated side. Lamina V contained an average of 2.2 and 5. I labelled cells on the intact and denervated sides, respectively. Very similar results were found in the rat that survived 7 days after peripheral nerve section. The lower number of cells in the latter two animals presumably reflects variability in the effectiveness of the colchicine treatment. Some sections from two deafferented rats were stained with an antiserum directed against the enkephalin octapeptide, enkephalin-Arg-Gly-Leu. Although this antiserum stains enkephalin perikarya in colcichine-treated animals, we found no differences in the number or intensity of labelled cells on the deafferented and unoperated sides of the cord. We previously reported that deafferentation results in a decrease in the density o t immunoreactive dynorphin terminals in the superficial dorsal horn and in lamina V of the sacral spinal cord of the cat [2]. This decrease was presumed to result from the loss of a population of primary afferent fibers that contain dynorphin. Since that study was not performed in colchicine-treated animals, no analysis of perikaryal dynorphin staining was reported. The present data illustrate that deafferentation leads to significant increase in both the number and in the intensity of staining of labelled dynorphin cells. Since we omitted Triton from the antibody solutions, there was minimal terminal staining and thus the effect of dorsal rhizotomy on the terminal staining pattern in colchieine-treated rats remains to be determined. The present data are reminiscent of several recent reports indicating that both dynorphin peptide levels (as measured by radioimmunoassay), and dynorphin message level increase in the lumbar spinal cord of both mono and polyarthritic rats [11 13, 18, 21]. The changes found in the arthritic rats were presumed to result from the increased spontaneous activity arising from the inflamed joints. Thus, increased nociceptive input appears to increase dynorphin message levels and this results in more peptide being synthesized. Interestingly, although noxious peripheral nerve stimulation increases the level of immunoreactive enkephalin in the spinal cord cerebrospinal fluid (CSF) [27], neither active inflammatory disease nor deafferentation increased tissue enkephalin levels to the extent that it did the levels of immunoreactive dynorphin. These more recent data contrast with the earlier report of a significant increase in the lumbar dorsal horn levels of Met-enkephalin in polyarthritic rats
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[6]. The latter studies, however, did not address dynorphin levels, nor the possibility of antibody cross-reactivity with dynorphin. The deafferentation-evoked increase in dynorphin cell body labelling found in the present study obviously did not result from increased nociceptive input. Deafferentation, however, results in significant increases in the spontaneous activity of dorsal horn neurons, presumably due to a loss of a tonic large fiber-mediated, indirect, inhibitory input [3, 16]. Conceivably, the increased spontaneous activity of dorsal horn neurons in deafferented animals contributes to the induction of the dynorphin message. That the increase is not secondary to anatomical deafferentation of the secondorder neuron is clear from the fact that comparable changes were found in rats that underwent sciatic nerve section. This latter injury is associated with significant chemical reorganization of the primary afferent terminal, including a significant loss of substance P staining, but the anatomical relationship with the second-order neuron is largely intact [25]. After peripheral nerve section there is also a decrease in large fibermediated inhibition which presumably would result in increased tonic activity of second-order spinal cord neurons [26]. Large increases in dynorphin peptide and message have also been found in the lumbar cord caudal to a crush injury of the thoracic cord [8]. Since intrathecal administration of dynorphin evokes a profound flaccid paralysis, it was proposed that the increased dynorphin that appears after spinal injury contributes to the prolonged paralysis of spinal injury. We only found changes in the dynorphin cells of the dorsal horn. Thus, it is unlikely that we are dealing with a population of neurons that directly interacts with spinal motoneurons. It is more likely that these neurons are involved in the transmission and or control of nociceptive messages. Since some dynorphin cells of the superficial dorsal horn project beyond the spinal cord, to the parabrachial nucleus [22] and to the nucleus of the solitary tract (Men6trey and Basbaum, unpublished observations) it will be of interest to examine brainstem sites for increases in terminal dynorphin immunoreactivity in deafferented and arthritic rats. In summary, this study has demonstrated that deafferentation, by either dorsal rhizotomy or peripheral nerve section, leads to a significant increase in the number of immunoreactive dynorphin neurons in the superficial dorsal horn and in lamina V of the spinal cord. These data indicate that increases in the central levels of dynorphin can result from a variety of injuries, not all of which are associated with increased nociceptive input. We thank Ms. Allison Gannon and Ms. Simona Ikeda for excellent photographic assistance. This work was supported by NIH Public Health Service Grants NS 14627 and 21445. I Basbaum, A.I., Functional analysis of the cytochemistry of the spinal dorsal horn, Adv. Pain Res. Ther.,9(1985) 149 175. 2 Basbaum, A.I., Cruz, L. and Weber, E.. Immunoreactive dynorphin in sacral primary afferent tibers of the cat, J. Neurosci., 6 (1986) 127 133. 3 Basbaum, A.I. and Wall, P.D., Chronic changes in the response of cells in adult cat dorsal horn following partial deafferentation: the appearance of responding cells in a previously non-responsive region. Brain Res.,116 (1976) 181 204. 4 Botticelli, LJ., Cox, B.M. and Goldstein, A., lmmunoreactive dynorphin in m a m m a l i a n spinal cord and dorsal root ganglia, Proc. Natl. Acad. Sci. USA, 78 (1981) 7783 7786.
130 5 Bowker, R.M., Westlund, K.N., Sullivan, M.C., Wilber, J.F. and Coulter, J.D., Transmitters of the raphe-spinal complex: immunocytochemical studies, Peptides, 3 (1982) 291-298. 6 Cesselin, F., Montastruc, J.L., Gros, C., Bourgoin, S. and Hamon, M., Met-enkephalin levels and opiate receptors in the spinal cord of chronic suffering rats, Brain Res., 191 (1980) 289-293. 7 Cruz, L. and Basbaum, A.I., Multiple opioid peptides and the modulation of pain: immunohistochemical analysis of dynorphin and enkephalin in the trigeminal nucleus caudalis and spinal cord of the cat, J. Comp. Neurol., 240 (1985) 331 348. 8 Faden, A.I., Molineaux, C.J., Rosenberg, J.G., Jacobs, T.P. and Cox, B.M., Increased dynorphin immunoreactivity in spinal cord after traumatic injury, Regul. Pept., 11 (1985) 35-41. 9 Glazer, E.J. and Basbaum, A.I., Opioid neurons and pain modulation: an ultrastructural analysis of enkephalin in cat superficial dorsal horn, Neuroscience, 10 (1983) 357 376. 10 H6kfelt, T., Terenius, L., Kuypers, H.G.J.M. and Dann, O., Evidence for enkephalin immunoreactive neurons in the medulla oblongata projecting to the spinal cord, Neurosci. Lett., 14 (1979) 55~0. 11 H611t, V., Haarman, I.,. Millan, M.J. and Herz, A., Prodynorphin gene expression is enhanced in the spinal cord of chronic arthritic rats, Neurosci. Lett., 73 (1987) 90-94. 12 Iadarola, M.J., Flores, C.M. and Naranjo, J.R., Increase in spinal cord dynorphin biosynthesis during peripheral inflammation: behavioral, neuropeptide and mRNA studies, Pain, Suppl. 4 (1987) 240. 13 Iadarola, M.J., Yang, H.-Y.T. and Costa, E., Increase in the content of spinal cord dynorphin during an experimentally-induced inflammation of the hindlimbs, Fed. Proc. Fed. Am. Soc. Exp. Biol., 44 (1985) 422. 14 Jessel, T.M. and Iversen, L.L., Opiate analgesics inhibit substance P release from rat trigeminal nucleus, Nature (Lond.), 268 (1977) 54%551. 15 Khachaturian, H., Watson, S.J., Lewis, M.E., Coy, D., Goldstein, A. and Akil, H., Dynorphin immunocytochemistry in the rat central nervous system, Peptides, 3 (1982) 941-954. 16 Lombard, M.C. and Larabi, Y., Electrophysiological study of cervical dorsal horn cells in partially deafferented rats, Adv. Pain Res. Ther., 5 (1983) 147-154. 17 Menbtrey, D. and Basbaum, A.I., The distribution of substance P, enkephalin and dynorphin immunoreactive neurons in the medulla of the rat and their contribution to bulbospinal pathways, Neuroscience, in press. 18 Millan, M.J., Millan, M.H., Czlonkowski, A., H611t, V., Pilcher, C.W.T., Colpaert, F.C. and Herz, A., A model of chronic pain in the rat: response of multiple opioid systems to adjuvant-induced arthritis, J. Neurosci., 6 (1986) 899-906. 19 Miller, K.E. and Seybold, V.S., Comparison of met-enkephalin-, dynorphin A-, and neurotensinimmunoreactive neurons in the cat and rat spinal cord: I. Lumbar cord, J. Comp. Neurol., 255 (1987) 293-304. 20 Ruda, M.A., Opiates and pain pathways: demonstration ofenkephalin synapses on dorsal horn projection neurons, Science, 215 (1982) 1523~-1524. 21 Ruda, M.A., ladarola, M. and Cohen, L., Immunocytochemical and in situ hybridization identify changes in neuronal dynorphin levels in a rat model of peripheral inflammation, Pain, Suppl. 4 (1987) 241. 22 Standaert, D.G., Watson, S.J., Houghten, R.A. and Saper, C.B., Opioid peptide immunoreactivity in spinal and trigeminal dorsal horn neurons projecting to the parabrachial nucleus in the rat, J. Neurosci., 6 (1986) 1220-1226. 23 Sweetnam, P.M., Neale, J.H., Barker, J.L. and Goldstein, A., Localization ofimmunoreactive dynorphin in neurons cultured from spinal cord and dorsal root ganglia, Proc. Natl. Acad. Sci. USA, 79 (1982) 6742~746. 24 Vincent, S.R., H6kfelt, T., Christensson, I. and Terenius, L., Dynorphin immunoreactive neurons in the central nervous system of the rat, Neurosci. Lett., 33 (1982) 185 190. 25 Wall, P.D., Fitzgerald, M. and Gibson, S.J., The response of rat spinal cord cells to unmyelinated afferents after peripheral nerve section and after changes in substance P levels, Neuroscience, 6 (I 981) 2205- 2215. 26 Woolf, C.J. and Wall, P.D., Chronic peripheral nerve section diminishes the primary afferent A-fiber mediated inhibition of dorsal horn neurons, Brain Res., 242 (1982) 77-85. 27 Yaksh, T.L. and Elde, R.P., Factors governing the release of methionine-enkephalin-like immunorcactivity from mesencephalon and spinal cord of the cat in vivo, J. Neurophysiol., 4 (1981) 1056-1075.
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NSL 05056
Increased staining of immunoreactive dynorphin cell bodies in the deafferented spinal cord of the rat Hee Jung Cho and Allan I. Basbaum Departments o[Anatomy and Physiology. University of Cali]brnia San Francisco. San Francisco. ('A 94143 (U.S.A.)
(Received 14 August 1987: Revised version received 22 September 1987; Accepted 29 September 1987} Key wor¢Lw Dynorphin: lmmunocytochemistry; Deafferentation: Dorsal horn; Pain: Rat
This study examined the distribution of immunoreactive dynorphin neurons in the lumbar dorsal horn of unilaterally deafferented, colchicine-treated rats. Ipsilateral to a multiple dorsal rhizolomy there was a significant increase both in the number and intensity of staining of dynorphin-immunoreactive cells in laminae I, outer II and V. A comparable change was seen in animals that were deafferented by sciatic nerve section. Enkephalin immunoreactivity was not altered under these conditions. These results indicate that many forms of injury, not all of which result in increased nociceptive input, can increase lhe level ofdynorphin in spinal cord neurons.
I m m u n o c y t o c h e m i c a l studies have identified significant differences in the spinal c o r d d i s t r i b u t i o n o f the two o p i o i d peptides, e n k e p h a l i n a n d d y n o r p h i n , M o s t n o t a bly, there is a relatively restricted d i s t r i b u t i o n o f d y n o r p h i n cell bodies, in l a m i n a e I, in the o u t e r p a r t o f the s u b s t a n t i a g e l a t i n o s a ( l a m i n a IIo) a n d in l a m i n a V [7, 15, 19, 24]. E n k e p h a l i n cell b o d i e s are m o r e widely d i s t r i b u t e d . T h e y are f o u n d in all layers o f the superficial d o r s a l horn, including the inner p a r t o f the s u b s t a n t i a gelatinosa and in l a m i n a lII, two regions typically a s s o c i a t e d with the processing o f inform a t i o n related to n o n - n o x i o u s inputs [1]. T h e r e is a c o r r e s p o n d i n g difference in the t e r m i n a l d i s t r i b u t i o n o f the two peptides. D y n o r p h i n t e r m i n a l s are c o n c e n t r a t e d in l a m i n a e l a n d V: e n k e p h a l i n t e r m i n a l s are m o r e widely d i s t r i b u t e d . Finally, some p r i m a r y afferents ( p a r t i c u l a r l y in the sacral spinal c o r d o f the cat) c o n t a i n d y n o r p h i n [2, 4, 23]. D o r s a l r h i z o t o m y decreases the t e r m i n a l d y n o r p h i n i m m u n o r e a c t i v i t y in the sacral spinal cord; as expected, there are m i n i m a l c h a n g e s in e n k e p h a l i n levels
[21 W i t h the exception o f a small b u l b o s p i n a l e n k e p h a l i n p r o j e c t i o n [5, 10, 17], the m a j o r i t y o f spinal e n k e p h a l i n arises f r o m spinal i n t e r n e u r o n s . T h e latter are h y p o t h e sized to exert a segmental a n t i n o c i c e p t i v e c o n t r o l via b o t h pre- a n d p o s t s y n a p t i c inhiCorrespmTdence: A.I. Basbaum, Departments of Anatomy and Physiology, University of California San
Francisco, San Francisco, CA 94143, U.S.A.
126
bition of spinal nociceptive neurons [14]. Anatomical studies, however, indicate that axodendritic enkephalin relationships predominate [9, 20]. To date, the relationship ofdynorphin terminals to primary afferent fibers has not been addressed. With a view to characterizing the synaptic relationship between degenerating primary afferent fibers and the intrinsic dynorphin cell population of the superficial dorsal horn, we have repeated the dorsal rhizotomy studies in rats that were treated with colchicine so that the local dynorphin cells of the dorsal horn could be visualized. This report describes a striking increase in dynorphin perikaryal immunoreactivity in the colchicine-treated, deafferented dorsal horn. Male rats were anesthetized with Nembutal (60 mg/kg) and a laminectomy was performed over the lumbosacral enlargement. At the caudal end of the enlargement the dura was incised and reflected laterally so as to expose the incoming dorsal roots. Dorsal roots Ls-S1 were then cut with iridectomy scissors. To increase the staining of dynorphin cell bodies, these studies were performed in colchicine-treated rats. After the roots were cut, a 10-/zl Hamilton syringe with a fixed 34-gauge needle was threaded under the dura, rostral to the rhizotomy, until the tip of the needle was located over the lumbar enlargement. Ten/A of a 20/zg/~tl solution of colchicine was injected slowly and then the needle was withdrawn. At no time did the needle come into contact with the cord surface. When the injection was completed, the animal was tilted head down to minimize caudal diffusion of the colchicine. After 3 min, a strip of moistened Gelfilm was placed over the exposed cord, the overlying muscle and skin were sutured and the rats returned to their cages. Twenty-four hours later the rats were reanesthetized and perfused intracardially with the following solutions: 100 ml of a 0.1 M phosphate-buffered saline wash and 400 ml of a fixative containing 0.1 M phosphate-buffered 4% paraformaldehyde, and in some cases 0.1% glutaraldehyde. The appropriate spinal cord segments were removed and the completeness of the rhizotomy verified under a dissecting microscope. After washing the tissue in phosphate-buffer (PB), fifty micron Vibratome sections through the lumbar enlargement were cut and collected into PB. The sections were then processed with the avitin-biotin method for localizing immunoreactive dynorphin. An antiserum directed against dynorphin B (a kind gift of Dr. Eckard Weber) was used at a dilution of i:3000. Some sections were osmicated and embedded in plastic for subsequent electron microscopic analysis. A detailed description of the synaptic relationship between immunoreactive dynorphin B cell bodies and terminals and degenerating primary afferent terminals will be described in another report. In two additional rats, the sciatic nerve was exposed at the sciatic notch and a ligature tied around it. At least 1 cm of the distal stump was cut and removed. One of the rats was administered colchicine intrathecally at the time of the peripheral denervation and was perfused 24 h later. The other rat was administered colchicine 6 days after the sciatic nerve section and was perfused 24 h later. It is rare to find immunoreactive dynorphin perikarya in the spinal cord of the normal rat. Twenty-four hours after colchicine treatment, however, there were large numbers of dynorphin cells labelled bilaterally in the lumbosacral enlargement. The
127
distribution of cells corresponded to those segments which were topically exposed to the colchicine. The majority of labelled cells were found in the superficial layers of the dorsal horn, laminae I and outer II. Fewer, but larger cells were found in the region of lamina V. Approximately equal numbers of labelled neurons were found on both sides of the cord; there was no detectable difference in the intensity of staining on the two sides. In rats that had undergone a multiple dorsal rhizotomy at the time of the colchicine administration, there was considerable asymmetry in the staining of the dynorphin cell bodies. Ipsilateral to the deafferentation there was a large increase in both the number and intensity of immunoreactive dynorphin cell bodies. The distribution of labelled cells, however, did not differ on the two sides of the cord. The increased perikaryal staining was found in the same region where labelled dynorphin cells are found in the unoperated animal, i.e. in the superficial dorsal horn and in the region of lamina V. Fig. 1 is taken from an osmicated, plastic embedded Vibratome section and illustrates that increased perikaryal staining can be seen across the mediolateral extent of the superficial dorsal horn, particularly in laminae I and outer II.
Fig. 1. lmmunoreactive dynorphin cells in the lumbar spinal cord of a unilaterally deafferented, colchicinetreated rat. The increased cell staining is evident in the superficial dorsal horn (laminae I and outer I1) of the deafferented (right) side of the cord (A). The areas outlined in the left and right boxes are magnified in B and C, respectively. Note that there is not only an increased number of immunoreactive dynorphin cells labelled on the deafferented side of the cord, but the density of the staining is also increased. Arrowheads in B and C identify immunoreactive dynorphin cell bodies. I, 11o and Ili identify lamina I, and the outer and inner portions of the substantia gelatinosa, respectively. Bars = 2 0 0 / l m in A and 50 t~m in B and C.
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To provide a quantitative estimate of the increased staining on the deafferented side, counts of labelled neurons were made in 15 sections through the lumbar enlargement of one rat. The sections from this rat were immunoreacted without Triton in the incubation media and were osmicated and embedded in plastic. This limited the terminal staining and made it much simpler to identify and count labelled cells. Cells were counted in two focal planes, corresponding to the two surfaces of the section. The average number of immunoreactive dynorphin cells in lamina I on the unoperated side was 24.6. On the deafferented side there was an average of 34.1 labelled cells. In lamina V, there was an average of 2.2 labelled cells on the unoperated side and 5.1 cells on the deafferented side. We also counted cells from unosmicated sections through the lumbar cord of the rats that underwent sciatic nerve section. Increased perikaryal staining was found in the dorsal horn ipsilateral to a sciatic nerve section. In the rat that survived 24 h after sciatic nerve section, there was an average of 13.8 labelled cells in laminae I and I1 per 50/zm Vibratome section on the unoperated side and 20.1 on the denervated side. Lamina V contained an average of 2.2 and 5. I labelled cells on the intact and denervated sides, respectively. Very similar results were found in the rat that survived 7 days after peripheral nerve section. The lower number of cells in the latter two animals presumably reflects variability in the effectiveness of the colchicine treatment. Some sections from two deafferented rats were stained with an antiserum directed against the enkephalin octapeptide, enkephalin-Arg-Gly-Leu. Although this antiserum stains enkephalin perikarya in colcichine-treated animals, we found no differences in the number or intensity of labelled cells on the deafferented and unoperated sides of the cord. We previously reported that deafferentation results in a decrease in the density o t immunoreactive dynorphin terminals in the superficial dorsal horn and in lamina V of the sacral spinal cord of the cat [2]. This decrease was presumed to result from the loss of a population of primary afferent fibers that contain dynorphin. Since that study was not performed in colchicine-treated animals, no analysis of perikaryal dynorphin staining was reported. The present data illustrate that deafferentation leads to significant increase in both the number and in the intensity of staining of labelled dynorphin cells. Since we omitted Triton from the antibody solutions, there was minimal terminal staining and thus the effect of dorsal rhizotomy on the terminal staining pattern in colchieine-treated rats remains to be determined. The present data are reminiscent of several recent reports indicating that both dynorphin peptide levels (as measured by radioimmunoassay), and dynorphin message level increase in the lumbar spinal cord of both mono and polyarthritic rats [11 13, 18, 21]. The changes found in the arthritic rats were presumed to result from the increased spontaneous activity arising from the inflamed joints. Thus, increased nociceptive input appears to increase dynorphin message levels and this results in more peptide being synthesized. Interestingly, although noxious peripheral nerve stimulation increases the level of immunoreactive enkephalin in the spinal cord cerebrospinal fluid (CSF) [27], neither active inflammatory disease nor deafferentation increased tissue enkephalin levels to the extent that it did the levels of immunoreactive dynorphin. These more recent data contrast with the earlier report of a significant increase in the lumbar dorsal horn levels of Met-enkephalin in polyarthritic rats
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[6]. The latter studies, however, did not address dynorphin levels, nor the possibility of antibody cross-reactivity with dynorphin. The deafferentation-evoked increase in dynorphin cell body labelling found in the present study obviously did not result from increased nociceptive input. Deafferentation, however, results in significant increases in the spontaneous activity of dorsal horn neurons, presumably due to a loss of a tonic large fiber-mediated, indirect, inhibitory input [3, 16]. Conceivably, the increased spontaneous activity of dorsal horn neurons in deafferented animals contributes to the induction of the dynorphin message. That the increase is not secondary to anatomical deafferentation of the secondorder neuron is clear from the fact that comparable changes were found in rats that underwent sciatic nerve section. This latter injury is associated with significant chemical reorganization of the primary afferent terminal, including a significant loss of substance P staining, but the anatomical relationship with the second-order neuron is largely intact [25]. After peripheral nerve section there is also a decrease in large fibermediated inhibition which presumably would result in increased tonic activity of second-order spinal cord neurons [26]. Large increases in dynorphin peptide and message have also been found in the lumbar cord caudal to a crush injury of the thoracic cord [8]. Since intrathecal administration of dynorphin evokes a profound flaccid paralysis, it was proposed that the increased dynorphin that appears after spinal injury contributes to the prolonged paralysis of spinal injury. We only found changes in the dynorphin cells of the dorsal horn. Thus, it is unlikely that we are dealing with a population of neurons that directly interacts with spinal motoneurons. It is more likely that these neurons are involved in the transmission and or control of nociceptive messages. Since some dynorphin cells of the superficial dorsal horn project beyond the spinal cord, to the parabrachial nucleus [22] and to the nucleus of the solitary tract (Men6trey and Basbaum, unpublished observations) it will be of interest to examine brainstem sites for increases in terminal dynorphin immunoreactivity in deafferented and arthritic rats. In summary, this study has demonstrated that deafferentation, by either dorsal rhizotomy or peripheral nerve section, leads to a significant increase in the number of immunoreactive dynorphin neurons in the superficial dorsal horn and in lamina V of the spinal cord. These data indicate that increases in the central levels of dynorphin can result from a variety of injuries, not all of which are associated with increased nociceptive input. We thank Ms. Allison Gannon and Ms. Simona Ikeda for excellent photographic assistance. This work was supported by NIH Public Health Service Grants NS 14627 and 21445. I Basbaum, A.I., Functional analysis of the cytochemistry of the spinal dorsal horn, Adv. Pain Res. Ther.,9(1985) 149 175. 2 Basbaum, A.I., Cruz, L. and Weber, E.. Immunoreactive dynorphin in sacral primary afferent tibers of the cat, J. Neurosci., 6 (1986) 127 133. 3 Basbaum, A.I. and Wall, P.D., Chronic changes in the response of cells in adult cat dorsal horn following partial deafferentation: the appearance of responding cells in a previously non-responsive region. Brain Res.,116 (1976) 181 204. 4 Botticelli, LJ., Cox, B.M. and Goldstein, A., lmmunoreactive dynorphin in m a m m a l i a n spinal cord and dorsal root ganglia, Proc. Natl. Acad. Sci. USA, 78 (1981) 7783 7786.
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