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Release and depletion of substance P by capsaicin in substantia gelatinosa studied with the antibody microprobe technique and immunohistochemistry
Neuropepfides (1992) 23, 161-167 0 Longman Group UK Ltd 1992
Release and Depletion of Substance P by Capsaicin in Substantia Gelatinosa Studied with the Antik )ody Microprobe Technique and Immunohistochemi stry Z-Q. ZHAO, H.-Q. YANG, K.-M. ZHANG, X.-X. ZHUANG Shanghai Brain Research Institute, (Reprint requests to ZQZ)
Chinese Academy
of Sciences,
Shanghai
200031, China
Abstract-Using an antibody microprobe technique, we have detected substance P release from the region of the substantia gelatinosa of the cat during the first, but not the second, 30 min of topical application of capsaicin (l-3%) to the tibia1 nerve. lmmunohistochemical analysis also showed that substance P-like immunoreactivity was markedly reduced in the superficial layer of the dorsal horn 30 min after application of capsaicin. These results indicate that substance P is released and then depleted from primary afferent central terminals following acute application of capsaicin to the peripheral sensory nerve.
Introduction Substance P (SP) is present in a proportion of small spinal ganglion neurons and numerous terminals in the superficial laminae of the spinal dorsal horn (l-4), which is known to be the major central site of termination of nociceptors (5). Substance P is released from activated primary afferent fibers in vitro (6-9), substance P has also been detected in the spinal cord perfusates following electrical stimulation of Ad and C fibers of the cat (lo), with a push-pull cannula inserted into the rabbit dorsal horn following noxious mechanical peripheral stimuli (1 I), and with microdialysis probe (12) or antibody microprobe (13, 14) inserted into the dorsal horn of
Date received 14 July 1992 Date accepted 15 July 1992
the cat during noxious cutaneous stimuli. It is, therefore, reasonable to suggest that substance P may be involved in the transmission and/or modulation of spinal nociceptive information. In addition to primary afferent fibers, some intrinsic intemeurons of the dorsal horn and descending fibers from supraspinal regions in the dorsal horn also contain substance P (1, 3). It remains a question whether release of substance P following peripheral stimuli originates directly from activated primary afferent fibers in the dorsal horn or whether it could reflect the excitation of substance P-containing intemeurons or descending fibers. Capsaicin, the pungent ingredient in peppers of the capsicum family, is known to act specifically on a subset of primary afferent sensory neurons with small diameter axons, i.e. C- and Ad-fibers (15). Capsaicin has been widely used experimentally to
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excite or damage selectively this subset of sensory neurons and to deplete substance P and other neuropeptides from primary afferent terminals (7, 16-l 9). Thus, Capsaicin is regarded as a useful pharmacological tool to study substance P-containing spinal ganglion neurons. The aim of the present work is to detect the process of release and depletion of substance P in intraspinal primary afferent terminals following acute topical application of capsaicin to the peripheral sensory nerve.
Methods The experiments were performed on 10 cats anaesthetized with sodium pentobarbitone (40 mg/kg i.p. initially). A superficial vein, a carotid artery, and the trachea were separately cannulated. The left and right tibia1 nerves were exposed for topical application of capsaicin. Following a lumbar laminectomy, the spinal cord was transected at thoracolumbar junction and covered with a thin layer Ringer-agar which was removed at sites of microprobe penetration. These sites were centered in the area where the maximal electrical response could be obtained on tibia1 nerve stimulation. Anaesthesia and neuromuscular paralysis were maintained throughout the experiment by continuous iv. infusion of sodium pentobarbitone (3 mgkgih) and gallamine triethiodide (4 mg/kgih). The body temperature, bloodpressure and end-tidal CO1 level were monitored continuously and kept within physiological limits. A detailed description of the preparation of antibody microprobes was written by Duggan et al (20). In brief, glass microelectrodes were coated with amino-silane polymer by immersion in a 10% solution of 3-aminopropyltriethoxy-silane (Aldrich) in toluene. Microelectrodes were then immersed in 2.5% glutaraldehyde followed by sequential incubations in solution of 2 mgml Protein A (Sigma) and a 1:400 dilution in PBS of a rabbit antiserum substance P (kindly provided by Academy of Chinese Traditional Medicine). Small openings were made in the spinal cord pia for the entry of microprobes and the dorsal surface of the cord was continually irrigated with warm artificial CSF. Two micromanipulators were used to introduce microprobes into the spinal cord. One
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microprobe was inserted into the spinal cord to the depth of 2 mm and other to the depth of 3 mm at 18” to the vertical. Some microprobes were filled with a 2% solution of Potamine sky blue in 1.2 M sodium acetate. Dye deposits were used to locate the position of the microprobe tips histologically. Microprobes were kept in position in the spinal cord for 30 min during each of the following procedures: (1) local immersion of the tibia1 nerve (10 mm) in silicone oil, (2) the first 30 min after local immersion of the tibia1 nerve in l-3% solution of capsaicin in silicone oil and (3) the second 30 min after application of capsaicin. After removal from the spinal cord, the antibody microprobes were washed in phosphate-buffered saline (PBS)-0.05% Tween solution for 15 min and incubtaed in PBS containing 1500-2000 cpm Bolton-Hunter L251-SP (Amersham) and bovine serum albumin (5 mgml) for 24 h. The microprobes were washed again and the tips broken off and placed on X-ray film for 24-72 h. In vitro assays were performed in parallel with all in vivo experiments. The microprobes were immersed in solutions of substance P (Sigma) with concentrations ranging from lo-lo to 1W M for 30 min. Preincubation in SP 1O-7M and 1O-5M suppressed the binding of L251-SP>50% and >90%, respectively. Thus non-specific binding to the microprobes should be less than 10% of total binding. Information from the supplier of the antibodies indicated negligible cross-reactivity with numerous other neuropeptides. Microprobe images were photographically enlarged and subjected to quantitative analysis with a video camera (RCA TC 7000), DT 2853/image Processing System and computer (AST Premium 286). A video camera scanned the autoradiograph of each microprobe and the optical density of this image was digitized to an arbitrary grey scale of 0 (absence of silver grains) to 255 (maximum grain density). This type of analysis enables averaging of the images from a specified group of microprobes. Digitized optical density values plotted with respect to distance from the tip were thus obtained for each microprobe, and mean image density scans were calculated for specified microprobes. Zones of reduced optical density (less radiolabel binding) were interpreted as regions of SP release which were indicated by the downward deviations of the slope. The com-
RELEASEAND DEPLETIONOF SP BY CAPSAICININ SUBSTANTIAGELATINOSA
puter calculated the t values for estimating the significance of observed differences. 30 min following topical application of 1% capsaicin on the right- and of silicone oil on the left-tibial nerve, the cats were perfused transcardially with 1000 ml normal saline followed by 2000 ml of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M PBS (PH 7.4). The spinal cords were postfixed in 50 ml fresh 0.1 M PBS perfusion solution for 1 h and then transferred to 0.1 PBS containing 30% solution of sucrose for 24 h at 4°C. 30 micrometer transverse serial sections were cut through L6-7 on a vibratome in 0.1 M PBS at 4°C. The section was incubated in primary antiserum against substance P (1: 1000) for 24-48 h at 4°C The sections were subsequently incubated in second antisera (goat antirabbit IgG, 1:30) for 1 h at room temperature, and peroxidase-antiperoxidase (PAP 1:200) for 1 h at room temperature. The peroxidase label was developed in 0.05% DAB for 10 min and then 4 drops of
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1% hydrogen peroxide were applied. Primary antisera, second antisera and PAP were diluted with 1% normal goat serum. All incubation steps were preceded by 2 washes in 0.1 M PBS for 5 min each.
Results Detection of SP release by antibody microprobes: A total of 150 microprobes were analysed in the course of the present study. Of these, 95 were used to detect substance P release in vivo. The remaining microprobes were used for parallel experiments in vitro. Release of substance P was measured before and after acute application of capsaicin to the tibia1 nerve. An example is shown in Figure 1. Photographic enlargements of 2 microprobe images have been superimposed on a same enlargement of a cross section of the spinal cord. Compared with microprobe B (without capsaicin), acute application
Fig. 1 Inhibition of binding of ‘*%SP to antibody microprobes with topical application of capsalcin to the left tibia1 nerve. Photographic enlargements of X-ray images of microprobes have been superimposed on a similarly enlarged section of the lumbar spinal cord. The position of tips of the microprobes was determined by the depth of microprobes inserted according to the reading of micromanipulator and dye deposits. Microprobe A was in the spinal cord during a 30 min application of capsaicin to the left tibia1 nerve. Microprobe B was in the spinal cord for 30 min without capsaicin.
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control
w
y1 800
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a
?i 5: k ”
:: $ ”
400.
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C
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Release of SP in the substantia gelatinosa by application of capsaicin on the tibia1 nerve. A) Control: graph of the mean image density scan (with S.E.M.) of 35 microprobes inserted 2 mm into the spinal cord during application of vehicle solution for 30 min. B) During application of capsaicin (the fist 30 min): graph of the mean image density scan of 27 microprobes. C) During 3040 min of capsaicin: graph of the mean image density scan of 33 microprobes. Ordinate: optical density is expressed as an arbitrary grey scale. Abscissa: depth in the spinal cord (mm), tips being at 2 mm. Fig. 2
of capsaicin (probe A) produced an intense zone of inhibition of binding of 1251-SPin the substantia gelatinosa of the dorsal horn, indicating the region of SP release. The mean image density scans by the video computer were obtained from 95 microprobes (Fig.2). Of these, 35 were controls (tibia1 nerve bathed in vehicle); 27 were inserted in the spinal cord during the first 30 min of application of capsaicin to the nerve; 33 were inserted during the second 30 min. When microprobes were kept in the spinal cord during the first 30 min of application of capsaicin, there was a zone of inhibition of binding of 1251-SP
centered 1.2 mm from the spinal cord surface (Fig. 2B), indicating SP release in this zone which corresponded to the SG region of the dorsal horn. However, microprobes inserted during administration of vehicle solution (Fig. 2A) and during the second 30 min of administration of capsaicin (Fig. 2C) showedno SP release. Comparisons have been made between the mean image scans of microprobes without and with application of capsaicin. Subtracting capsaicin from control gave a record in Fig. 3 obtained from Fig. 2B and 2A, showing that difference was significant (p < 0.05). Immunohistochemical observations: Following
RELEASE
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1000 DIFFERENCE
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0 t value
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-1000 Fig. 3 Capsaicin-induced release of substance P in the region of substantia gelatinosa. A) Comparison between graphs of control (35 microprobes) and capsaicin (27 microprobes) tests, showing that capsaicin includes a remarkable depression at a depth of 1.2 mm from surface of the spinal cord. B) The differences between the two graphs and t-value calculated for these differences. The broken line is the t-value indicating significance @ = 0.05).
topical application of capsaicin and vehicle, respectively, on the right- and left-tibia1 nerves for 30 min, there was a significant difference in the concentrations of SP-like immunoreactivity (SP-li) in the two dorsal horns of the lumbar spinal cord. Low magnification light microscopy of the spinal cord section revealed the heaviest concentration of SP-Li in lam-
inae I - II and moderate density in lamina X of the right dorsal horn (Fig. 4). In contrast, acute treatment of the left tibia1 nerve by capsaicin produced a profound loss of SP-li in the left dorsal horn. There was no effect of capsaicin on the concentration of SP-Ii in the thoracic spinal cord. The regions containing the superficial dorsal horns in transverse sec-
Fig. 4 A photograph from the screen of the video monitor shows depletion of SP-li in the superficial dorsal horn of the lumbar spinal cord. Right side: 30 min after application of oil to the left tibia1 nerve, showing dense distribution of SP-li in the dorsal horn. Left side : 30 min after application of capsaicin to the right tibia1 nerve. Numbers represent arbitrary grey scale.
166 tions from 3 cats were quantitatively analysed with an automated image analysis system. The concentration of SP-li was reduced by 20% following acute application of capsaicin.
Discussion The capsaicin evoked release of substance P in the spinal cord has been demonstrated both in vivo and in vitro, by previous workers (7,8, 10, 11, 16). The nerve elements which liberate substance P following peripheral noxious stimulation remained to be determined. As mentioned above, descending fibers, intrinsic interneurons and small primary sensory neurons all could be candidates. If substance P is a mediator of spinal nociception, SP release must be confined to the endings of the primary unmyelinated fibers. Since capsaicin seems to depolarizeprimary sensory unmyelinated fibers but not on interneurons in the spinal cord (2 1,22) it was used to determine whether unmyelinated fibers release substance P. Using the antibody microprobe, we were indeed able to localize substance P release to the region of the substantia gelatinosa where primary unmyelinated afferent fibers terminate following acute application of capsaicin to the peripheral nerve. This is consistent with the previous results obtained using noxious stimuli other than capsaicin (13, 14). Thus, SP release in the substantia gelatinosa induced by noxious stimuli is, mainly from primary afferent terminals. High level transection of the spinal cord in the present experiments excluded the possibility of SP release from terminals of descending fibers. The previous observations that depletion ofbulbospinal serotonin and SP stores by intrathecal5.6~dihyroxytryptamine had no effect on SP release by intrathecal capsaicin (23) and that SP release in the substantia gelatinosa by noxious peripheral stimuli was not altered by blocking spinal conduction at the first lumbar segment by cooling the spinal cord (24) both lend support to the results of our present investigation. Further support of our experiment comes from the observation that substance P release by perfusion of the spinal cord with capsaicin was not diminished by intrathecal kainic acid, which selectively damages cell bodies without affecting presynaptic processes (25). Our recent __ studies using antibody microprobes also showed that
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no change of nerve stimuli-induced SP release in the SG was observed after local application of kainic acid on the small area of the spinal dorsal surface where the antibody microprobes were inserted, indicating that SP release was not from intrinsic neurons. In addition, after destruction of primary unmyelinated fibers by neonatal capsaicin treatment, high concentrations of K*, but not capsaicin, could still produce SP release as measured in the perf&ate of slices of the rat spinal cord, suggesting that the SP release in this case was from intrinsic neurons (26). Taken together, the experimental evidence shows that SP release produced by acute perineural treatment of capsaicin and other noxious peripheral stimuli is indeed derived from the primary afferent terminals in the spinal cord. The fact as shown in the present study that SP release in the substantia gelatinosa could be detected only in the first 30 min of capsaicin treatment, but not afterwards seems to agree with the findings by Go and Yaksh who demonstrated that after pefision of the spinal cord with capsaicin nerve stimulation failed to evoke SP release (10). They assumed that this desensitization was not due to a general depletion of SP because there was no decline in the levels of spinal SP-li immediately after the capsaicin treatment. This interpretation, however, is questionable, since in their experiments SP release by capsaicin was measured from perfusate of all segments of the spinal cord, not from one precise location. Although the content of SP in primary terminals of the sciatic nerve might be markedly reduced by a high concentration of capsaicin in the lumbar segments, a local effect of capsaicin on primary terminals of the sciatic nerve might be masked by the level of release of SP-li in the perfusate of all segments of the spinal cord. To answer this question directly, immunohistochemistry is helpful. If the failure of SP release after capsaicin reflects an acute depletion of SP from primary afferent terminals, the density of SP-li in the substantia gelatinosa should be reduced. This was indeed the case. However, since long-lasting inhibition of voltage-activated calcium channels were produced by capsaicin in dorsal root neuron in vitro (27), inhibition of SP release may partially involve in this mechanism. It is interesting to note that the substance P is released mainly in the substantia gelatinosa where
RELEASE
AND DEPLETION
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IN SUBSTANTIA
substance P, opioid peptides (28), opioid receptors (29) and primary unmyelinated afferent fibers conveying nociceptive signals (5) intermingle and interact closely. Physiologically, the substantia gelatinosa probably plays a major role in control of nociceptive transmission at the spinal level. Acknowledgements We thank Professor Chang Hsiang-Tung for his helpful criticism and revision of the manuscript, and Professor Bao Xuan for her guiding in immunohistochemical experiments. This project supported by the National Natural Science Foundation of China.
References 1 Baber, R. P., Vaughn, J. E., Skemmon, J. R., Salvterra, P. M., Roberts, T. and Leeman, S. E. (1979). The origin, distribution and synaptic relationships of substance P axons in rat spinalcord. JoumalofComparativeNeurology 184: 33 l-352. 2. Ju, G., H&felt, T., Brodin, E. et al. (1987). Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells. Cell Tissue Research 247: 41743 1. 3. HBkfelt, T., Ljungdahl, A., Steinbusch, H. et al. (1978). Immunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system. Neuroscience 3: 517-538. 4. Riberiro-Da-Silva, A., Tagari, P. and Cuello, A. C. (1989). characterization Morphological of substance P-like immunoreactive glormeruli in the superficial dorsal horn of the rat spinal cord and trigeminal subnucleus caudalis: A quantitative study. Journal Comparative Neurology 28 1: 497-5 15. 5. Sugiura, Y., Lee, C. L. and Perl, E. R. (1986). Central projections of identified, unmyelinated (C) afferent fibers innervating mammalian skin. Science 234: 358-361. 6. Busies, A. and Lembeck, F. (1981). In vitro release of substance P from spinal cord slices by capsaicin congeners. European Journal of Pharmacology 7 1: 7 l-77. 7. Fitzgerald, M. (1983). Capsaicin and sensory neurones-a review. Pain 15: 109-130. 8. Jessel, T. M., Tsunoo, A., Kanazawa, I. and Otsuka, M. (1979).Substance P deletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons, Brain Research 168: 247-256. 9. Otsuka, M. and Konishi, S. (1976). Release of substance Plike immunoreactivity from isolated spinal cord of newborn rabbit. Nature 264: 83-84. 10. Go, V. L. W. and Yaksh, T. L. (1987). Release of substance P from the cat spinal cord. Journal of Physiology 391: 141-167. 11. Kuraishi, Y., Hirota, N., Sato, Y., Hino, Y., Satoh, M. and Takagi, H. (1985). Evidence that substance P and somatostatin transmit separate information related to pain in the spinal dorsal horn. Brain Research 325: 294298. 12. Brodin, E., Linderoth, B., Gazelius, B. and Ugerstedt, U. (1987). In vivo release of substance P in cat dorsal horn studied with microdialysis. Neuroscience Letters 76: 357-362.
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13. Duggan, A. W., Hentry, I. A., Morton, C. R., Hutchison, W. D. and Zhao, Z. Q. (1988). Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat. Brain Research 451: 261-273. 14. Duggan, A. W., Morton, C. R., Zhao, Z. Q. andHentry, I. A. (1987) Noxious heating of the skin releases immunoreactive substance P the substantia gelatinosa of the cat: a study with antibody microprobes. Brain Research 403: 345-349. 1c IJ. Gamse, C. T. A., Molnar, A. and Lembeck, F. Substance P release from spinal cord slices by capsaocon. Life Science 25: 629636. 16. Aimone, L. D. and Yaksh, T. L. (1989). Gpioid modulation of capsaicin-evoked release of substance P from rat spinal cord in vivo. Peptides 10: 1127-113 1. 17. Buck, S. A. and Burks, T. F. (1986). The neuropharmacology of capsaicin: Review of some recent observations. Pharmacological Review. 38: 179-226. 18. Jancso, G. and Lawson, S. N. (1990). Transganglionic degeneration of capsaicm-sensitive C-fiber primary afferent terminals. Neuroscience 39: 501-511. 19. Theriault, E., Otuka, M. and Jessel, T. (1979). Capsaicinevoked release of substance P from primary sensory neuron calcium-dependent action potentials. Brain Research 170: 209-213. 20. Duggan, A. W., Hentry, I. A., Green, J. L., Morton, C. R. and Hutchison, W. D. (1988). The preparation and use of antibody microprobes. Journal of neuroscience Methods 23: 241-247. 21. Nagy, J. I. (1982). Capsaicin’s action on the nervous system. TINS 5: 362-365. 22. Ritter. S. and Dinh, T. T. (1988). Capsaicin-induced neuronal degenerations : Silver impregnation of cell bodies, axon and terminals in the central nervous system of the adult rat. Journal Comparative Neurology 271: 79-90. 23. Jessel. T. M. (1982). Substance P in the nervous system. In: Substance P in the nervous System. Ciba Foundation Symposium, 9 1: 225-248, London, Pittman. 24. Duggan, A. W., Morton, C. R., Hutchison, W. D. and Hentry, I. A. (1988). Absence of tonic supraspinal control of substance P release in the substantia gelatinosa of the anaesthetized cat. Experimental Brain Research 71: 597-602. 25. Jhamandas, K., Yaksh, T. L., Harty, G., Szolcsanyi, J. and Go, V. L. W. (1984). Action of intrathecal capsaicin and its structural analogues on the content and release of spinal substance P: Selectivity of action and relationship to analgesia. Brain Research 306: 2 15-225. 26. Pohl, M., Mauborgne, A., Bourgoin, S., Benoliel, J. J., Hamon, M. and Cesselin, F. (1989). Neonatal capsaicin treatment abolishes the modulations by opioids of substance P release from rat spinal cord slices. Neuroscience Letters 96: 102-107. 27. Docherty, R. J., Robertson, B. and Bevan, S. (1991). Capsaicin causes prolonged inhibition of voltage-activated calcium currents in adult rat dorsal root ganglion neurons in culture. Neuroscience 40: 5 13-52 1. 28. Hiikfelt, T., Ljungdahl, A., Terenius, T., Elde, R. andNilsson, G. (1977). Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: Enkephalm and substance P. Proceedings of the National Academy of Sciences USA 74: 3081-3085. 29. Lamotte, C., Pert, C. B. and Synder, S. H. (1976). Opiate receptor binding in primate spinal cord: Distribution and changes after dorsal root section. Brain Research 112: 407-412.
Release and Depletion of Substance P by Capsaicin in Substantia Gelatinosa Studied with the Antik )ody Microprobe Technique and Immunohistochemi stry Z-Q. ZHAO, H.-Q. YANG, K.-M. ZHANG, X.-X. ZHUANG Shanghai Brain Research Institute, (Reprint requests to ZQZ)
Chinese Academy
of Sciences,
Shanghai
200031, China
Abstract-Using an antibody microprobe technique, we have detected substance P release from the region of the substantia gelatinosa of the cat during the first, but not the second, 30 min of topical application of capsaicin (l-3%) to the tibia1 nerve. lmmunohistochemical analysis also showed that substance P-like immunoreactivity was markedly reduced in the superficial layer of the dorsal horn 30 min after application of capsaicin. These results indicate that substance P is released and then depleted from primary afferent central terminals following acute application of capsaicin to the peripheral sensory nerve.
Introduction Substance P (SP) is present in a proportion of small spinal ganglion neurons and numerous terminals in the superficial laminae of the spinal dorsal horn (l-4), which is known to be the major central site of termination of nociceptors (5). Substance P is released from activated primary afferent fibers in vitro (6-9), substance P has also been detected in the spinal cord perfusates following electrical stimulation of Ad and C fibers of the cat (lo), with a push-pull cannula inserted into the rabbit dorsal horn following noxious mechanical peripheral stimuli (1 I), and with microdialysis probe (12) or antibody microprobe (13, 14) inserted into the dorsal horn of
Date received 14 July 1992 Date accepted 15 July 1992
the cat during noxious cutaneous stimuli. It is, therefore, reasonable to suggest that substance P may be involved in the transmission and/or modulation of spinal nociceptive information. In addition to primary afferent fibers, some intrinsic intemeurons of the dorsal horn and descending fibers from supraspinal regions in the dorsal horn also contain substance P (1, 3). It remains a question whether release of substance P following peripheral stimuli originates directly from activated primary afferent fibers in the dorsal horn or whether it could reflect the excitation of substance P-containing intemeurons or descending fibers. Capsaicin, the pungent ingredient in peppers of the capsicum family, is known to act specifically on a subset of primary afferent sensory neurons with small diameter axons, i.e. C- and Ad-fibers (15). Capsaicin has been widely used experimentally to
161
162
excite or damage selectively this subset of sensory neurons and to deplete substance P and other neuropeptides from primary afferent terminals (7, 16-l 9). Thus, Capsaicin is regarded as a useful pharmacological tool to study substance P-containing spinal ganglion neurons. The aim of the present work is to detect the process of release and depletion of substance P in intraspinal primary afferent terminals following acute topical application of capsaicin to the peripheral sensory nerve.
Methods The experiments were performed on 10 cats anaesthetized with sodium pentobarbitone (40 mg/kg i.p. initially). A superficial vein, a carotid artery, and the trachea were separately cannulated. The left and right tibia1 nerves were exposed for topical application of capsaicin. Following a lumbar laminectomy, the spinal cord was transected at thoracolumbar junction and covered with a thin layer Ringer-agar which was removed at sites of microprobe penetration. These sites were centered in the area where the maximal electrical response could be obtained on tibia1 nerve stimulation. Anaesthesia and neuromuscular paralysis were maintained throughout the experiment by continuous iv. infusion of sodium pentobarbitone (3 mgkgih) and gallamine triethiodide (4 mg/kgih). The body temperature, bloodpressure and end-tidal CO1 level were monitored continuously and kept within physiological limits. A detailed description of the preparation of antibody microprobes was written by Duggan et al (20). In brief, glass microelectrodes were coated with amino-silane polymer by immersion in a 10% solution of 3-aminopropyltriethoxy-silane (Aldrich) in toluene. Microelectrodes were then immersed in 2.5% glutaraldehyde followed by sequential incubations in solution of 2 mgml Protein A (Sigma) and a 1:400 dilution in PBS of a rabbit antiserum substance P (kindly provided by Academy of Chinese Traditional Medicine). Small openings were made in the spinal cord pia for the entry of microprobes and the dorsal surface of the cord was continually irrigated with warm artificial CSF. Two micromanipulators were used to introduce microprobes into the spinal cord. One
NEUROPEPTIDES
microprobe was inserted into the spinal cord to the depth of 2 mm and other to the depth of 3 mm at 18” to the vertical. Some microprobes were filled with a 2% solution of Potamine sky blue in 1.2 M sodium acetate. Dye deposits were used to locate the position of the microprobe tips histologically. Microprobes were kept in position in the spinal cord for 30 min during each of the following procedures: (1) local immersion of the tibia1 nerve (10 mm) in silicone oil, (2) the first 30 min after local immersion of the tibia1 nerve in l-3% solution of capsaicin in silicone oil and (3) the second 30 min after application of capsaicin. After removal from the spinal cord, the antibody microprobes were washed in phosphate-buffered saline (PBS)-0.05% Tween solution for 15 min and incubtaed in PBS containing 1500-2000 cpm Bolton-Hunter L251-SP (Amersham) and bovine serum albumin (5 mgml) for 24 h. The microprobes were washed again and the tips broken off and placed on X-ray film for 24-72 h. In vitro assays were performed in parallel with all in vivo experiments. The microprobes were immersed in solutions of substance P (Sigma) with concentrations ranging from lo-lo to 1W M for 30 min. Preincubation in SP 1O-7M and 1O-5M suppressed the binding of L251-SP>50% and >90%, respectively. Thus non-specific binding to the microprobes should be less than 10% of total binding. Information from the supplier of the antibodies indicated negligible cross-reactivity with numerous other neuropeptides. Microprobe images were photographically enlarged and subjected to quantitative analysis with a video camera (RCA TC 7000), DT 2853/image Processing System and computer (AST Premium 286). A video camera scanned the autoradiograph of each microprobe and the optical density of this image was digitized to an arbitrary grey scale of 0 (absence of silver grains) to 255 (maximum grain density). This type of analysis enables averaging of the images from a specified group of microprobes. Digitized optical density values plotted with respect to distance from the tip were thus obtained for each microprobe, and mean image density scans were calculated for specified microprobes. Zones of reduced optical density (less radiolabel binding) were interpreted as regions of SP release which were indicated by the downward deviations of the slope. The com-
RELEASEAND DEPLETIONOF SP BY CAPSAICININ SUBSTANTIAGELATINOSA
puter calculated the t values for estimating the significance of observed differences. 30 min following topical application of 1% capsaicin on the right- and of silicone oil on the left-tibial nerve, the cats were perfused transcardially with 1000 ml normal saline followed by 2000 ml of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M PBS (PH 7.4). The spinal cords were postfixed in 50 ml fresh 0.1 M PBS perfusion solution for 1 h and then transferred to 0.1 PBS containing 30% solution of sucrose for 24 h at 4°C. 30 micrometer transverse serial sections were cut through L6-7 on a vibratome in 0.1 M PBS at 4°C. The section was incubated in primary antiserum against substance P (1: 1000) for 24-48 h at 4°C The sections were subsequently incubated in second antisera (goat antirabbit IgG, 1:30) for 1 h at room temperature, and peroxidase-antiperoxidase (PAP 1:200) for 1 h at room temperature. The peroxidase label was developed in 0.05% DAB for 10 min and then 4 drops of
163
1% hydrogen peroxide were applied. Primary antisera, second antisera and PAP were diluted with 1% normal goat serum. All incubation steps were preceded by 2 washes in 0.1 M PBS for 5 min each.
Results Detection of SP release by antibody microprobes: A total of 150 microprobes were analysed in the course of the present study. Of these, 95 were used to detect substance P release in vivo. The remaining microprobes were used for parallel experiments in vitro. Release of substance P was measured before and after acute application of capsaicin to the tibia1 nerve. An example is shown in Figure 1. Photographic enlargements of 2 microprobe images have been superimposed on a same enlargement of a cross section of the spinal cord. Compared with microprobe B (without capsaicin), acute application
Fig. 1 Inhibition of binding of ‘*%SP to antibody microprobes with topical application of capsalcin to the left tibia1 nerve. Photographic enlargements of X-ray images of microprobes have been superimposed on a similarly enlarged section of the lumbar spinal cord. The position of tips of the microprobes was determined by the depth of microprobes inserted according to the reading of micromanipulator and dye deposits. Microprobe A was in the spinal cord during a 30 min application of capsaicin to the left tibia1 nerve. Microprobe B was in the spinal cord for 30 min without capsaicin.
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164
control
w
y1 800
800
a
?i 5: k ”
:: $ ”
400.
0
0
400
0
I 2
1
1
2
DEPTH IN SPINAL CORD bnm)
DEPTH IN SPINAL CORD (mm1
C
1200
01
30 mln after capraioin
4
0
2
1 DEPTH IN SPINAL CORD
lmml
Release of SP in the substantia gelatinosa by application of capsaicin on the tibia1 nerve. A) Control: graph of the mean image density scan (with S.E.M.) of 35 microprobes inserted 2 mm into the spinal cord during application of vehicle solution for 30 min. B) During application of capsaicin (the fist 30 min): graph of the mean image density scan of 27 microprobes. C) During 3040 min of capsaicin: graph of the mean image density scan of 33 microprobes. Ordinate: optical density is expressed as an arbitrary grey scale. Abscissa: depth in the spinal cord (mm), tips being at 2 mm. Fig. 2
of capsaicin (probe A) produced an intense zone of inhibition of binding of 1251-SPin the substantia gelatinosa of the dorsal horn, indicating the region of SP release. The mean image density scans by the video computer were obtained from 95 microprobes (Fig.2). Of these, 35 were controls (tibia1 nerve bathed in vehicle); 27 were inserted in the spinal cord during the first 30 min of application of capsaicin to the nerve; 33 were inserted during the second 30 min. When microprobes were kept in the spinal cord during the first 30 min of application of capsaicin, there was a zone of inhibition of binding of 1251-SP
centered 1.2 mm from the spinal cord surface (Fig. 2B), indicating SP release in this zone which corresponded to the SG region of the dorsal horn. However, microprobes inserted during administration of vehicle solution (Fig. 2A) and during the second 30 min of administration of capsaicin (Fig. 2C) showedno SP release. Comparisons have been made between the mean image scans of microprobes without and with application of capsaicin. Subtracting capsaicin from control gave a record in Fig. 3 obtained from Fig. 2B and 2A, showing that difference was significant (p < 0.05). Immunohistochemical observations: Following
RELEASE
AND DEPLETION
OF SP BY CAPSAICIN
IN SUBSTANTIA
165
GELATINOSA
1000 DIFFERENCE
500
1
2mm
0 t value
DEPTH IN SPINAL CORD -500
-1000 Fig. 3 Capsaicin-induced release of substance P in the region of substantia gelatinosa. A) Comparison between graphs of control (35 microprobes) and capsaicin (27 microprobes) tests, showing that capsaicin includes a remarkable depression at a depth of 1.2 mm from surface of the spinal cord. B) The differences between the two graphs and t-value calculated for these differences. The broken line is the t-value indicating significance @ = 0.05).
topical application of capsaicin and vehicle, respectively, on the right- and left-tibia1 nerves for 30 min, there was a significant difference in the concentrations of SP-like immunoreactivity (SP-li) in the two dorsal horns of the lumbar spinal cord. Low magnification light microscopy of the spinal cord section revealed the heaviest concentration of SP-Li in lam-
inae I - II and moderate density in lamina X of the right dorsal horn (Fig. 4). In contrast, acute treatment of the left tibia1 nerve by capsaicin produced a profound loss of SP-li in the left dorsal horn. There was no effect of capsaicin on the concentration of SP-Ii in the thoracic spinal cord. The regions containing the superficial dorsal horns in transverse sec-
Fig. 4 A photograph from the screen of the video monitor shows depletion of SP-li in the superficial dorsal horn of the lumbar spinal cord. Right side: 30 min after application of oil to the left tibia1 nerve, showing dense distribution of SP-li in the dorsal horn. Left side : 30 min after application of capsaicin to the right tibia1 nerve. Numbers represent arbitrary grey scale.
166 tions from 3 cats were quantitatively analysed with an automated image analysis system. The concentration of SP-li was reduced by 20% following acute application of capsaicin.
Discussion The capsaicin evoked release of substance P in the spinal cord has been demonstrated both in vivo and in vitro, by previous workers (7,8, 10, 11, 16). The nerve elements which liberate substance P following peripheral noxious stimulation remained to be determined. As mentioned above, descending fibers, intrinsic interneurons and small primary sensory neurons all could be candidates. If substance P is a mediator of spinal nociception, SP release must be confined to the endings of the primary unmyelinated fibers. Since capsaicin seems to depolarizeprimary sensory unmyelinated fibers but not on interneurons in the spinal cord (2 1,22) it was used to determine whether unmyelinated fibers release substance P. Using the antibody microprobe, we were indeed able to localize substance P release to the region of the substantia gelatinosa where primary unmyelinated afferent fibers terminate following acute application of capsaicin to the peripheral nerve. This is consistent with the previous results obtained using noxious stimuli other than capsaicin (13, 14). Thus, SP release in the substantia gelatinosa induced by noxious stimuli is, mainly from primary afferent terminals. High level transection of the spinal cord in the present experiments excluded the possibility of SP release from terminals of descending fibers. The previous observations that depletion ofbulbospinal serotonin and SP stores by intrathecal5.6~dihyroxytryptamine had no effect on SP release by intrathecal capsaicin (23) and that SP release in the substantia gelatinosa by noxious peripheral stimuli was not altered by blocking spinal conduction at the first lumbar segment by cooling the spinal cord (24) both lend support to the results of our present investigation. Further support of our experiment comes from the observation that substance P release by perfusion of the spinal cord with capsaicin was not diminished by intrathecal kainic acid, which selectively damages cell bodies without affecting presynaptic processes (25). Our recent __ studies using antibody microprobes also showed that
NEUROPEFTIDES
no change of nerve stimuli-induced SP release in the SG was observed after local application of kainic acid on the small area of the spinal dorsal surface where the antibody microprobes were inserted, indicating that SP release was not from intrinsic neurons. In addition, after destruction of primary unmyelinated fibers by neonatal capsaicin treatment, high concentrations of K*, but not capsaicin, could still produce SP release as measured in the perf&ate of slices of the rat spinal cord, suggesting that the SP release in this case was from intrinsic neurons (26). Taken together, the experimental evidence shows that SP release produced by acute perineural treatment of capsaicin and other noxious peripheral stimuli is indeed derived from the primary afferent terminals in the spinal cord. The fact as shown in the present study that SP release in the substantia gelatinosa could be detected only in the first 30 min of capsaicin treatment, but not afterwards seems to agree with the findings by Go and Yaksh who demonstrated that after pefision of the spinal cord with capsaicin nerve stimulation failed to evoke SP release (10). They assumed that this desensitization was not due to a general depletion of SP because there was no decline in the levels of spinal SP-li immediately after the capsaicin treatment. This interpretation, however, is questionable, since in their experiments SP release by capsaicin was measured from perfusate of all segments of the spinal cord, not from one precise location. Although the content of SP in primary terminals of the sciatic nerve might be markedly reduced by a high concentration of capsaicin in the lumbar segments, a local effect of capsaicin on primary terminals of the sciatic nerve might be masked by the level of release of SP-li in the perfusate of all segments of the spinal cord. To answer this question directly, immunohistochemistry is helpful. If the failure of SP release after capsaicin reflects an acute depletion of SP from primary afferent terminals, the density of SP-li in the substantia gelatinosa should be reduced. This was indeed the case. However, since long-lasting inhibition of voltage-activated calcium channels were produced by capsaicin in dorsal root neuron in vitro (27), inhibition of SP release may partially involve in this mechanism. It is interesting to note that the substance P is released mainly in the substantia gelatinosa where
RELEASE
AND DEPLETION
OF SP BY CAPSAICIN
IN SUBSTANTIA
substance P, opioid peptides (28), opioid receptors (29) and primary unmyelinated afferent fibers conveying nociceptive signals (5) intermingle and interact closely. Physiologically, the substantia gelatinosa probably plays a major role in control of nociceptive transmission at the spinal level. Acknowledgements We thank Professor Chang Hsiang-Tung for his helpful criticism and revision of the manuscript, and Professor Bao Xuan for her guiding in immunohistochemical experiments. This project supported by the National Natural Science Foundation of China.
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