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Release of substance P from the spinal dorsal horn is enhanced in polyarthritic rats
Neuroscience Letters, 74 (1987) 315 319 Elsevier ScientificPublishers Ireland Ltd.
315
NSL 04456
Release of substance P from the spinal dorsal horn is enhanced in polyarthritic rats Ryoya Oku, Masamichi Satoh and Hiroshi Takagi Department ~[ Pharmacology, Faculty ( f Pharmaceutical Sciences, Kyoto University, Kvoto ~Japan) (Received 14 October 1986; Revised version received 7 November 1986: Accepted 10 November 1986} Key wordsv Substance P: Spinal dorsal horn; Release; Chronic pain; Polyarthritis; Rat Using in situ spinal dorsal horn perfusing method in decerebrated rats, we measured the release oF immunoreactive substance P (iSP) in polyarthritic rats. The spontaneous release of iSP from the dorsal horn in the polyarthritic rats was significantlyaccelerated over that in control non-inflamed rats. Passive movements of the inflamed ankle joint evoked a significant increase in the release of iSP, while a similar movement of the non-inflamedjoint led to no such increase. These results suggest that the facilitated release of iSP from the primary afferent terminals in the spinal dorsal horn in polyarthrific rats possibly plays a role in transmission of chronic pain of inflamedjoints.
Substance P (SP) is present in small diameter p r i m a r y sensory n e u r o n s [6] ternaihating in thc outer l a m i n a e of the spinal dorsal horn [5]. l o n t o p h o r e t i c a l l y applied SP has an excitatory action on some dorsal horn n e u r o n s that tire also excited by noxious stimuli [4, 10]. Moreover, intrathecal injection of the peptide elicits aversive b e h a v i o r in l a b o r a t o r y a n i m a l s [1 1]. Kuraishi et al. [8] used an in situ perfusion technique and ff)und a m a r k e d increase in release of i m m u n o r e a c t i v e SP (iSP) from the rabbit spinal dorsal horn d u r i n g acute noxious mechanical stimuli. These findings suggested that SP in the p r i m a r y sensory afferent participates in acute mechanical nociception. However, little is k n o w n of the i n v o l v e m e n t of the SP n e u r o n s in the spinal dorsal horn in chronic nociception. Thus, we directly cxamined a change in the release of" iSP from the l u m b a r dorsal horn d u r i n g chronic pain using polyarthritic rats as a reasonable model of chronic pain [I, 2, 9]. Polyarthritis was c o n v e n t i o n a l l y p r o d u c e d in male Sprague Dawley rats (220-280 g) by i n o c u l a t i n g a 0.08 ml s u s p e n s i o n of heat-killed Mycohacterium tuberculosis H 3 7 R A in paraffin oil (10 mg/ml) i n t r a d e r m a l l y into the footpad of the left hind paw. Two to 3 weeks later, the polyarthritic rats with severe swelling (foot volume, > 5.5 cm3; that of control rat is a b o u t 2.1 cm 3) were selected a n d used in the following studies. D u r i n g this period, the nociceptive thresholds in the hind paw pressure test were Correspondence." M. Satoh, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan. 0304-3940,'87:$ 03.50 @ 1987 ElsevierScientificPublishers Ireland Ltd.
316
A
B PUSH
P<0.05 vs. pre-stim release
n=3
PUU. 400
02
01
•
,
27G needle
~r
'5
space
200
~ 1oo i
0.8~ 1
~ 4
Pinch
or~)
3 Joint move.
4
Pinch
Fig. I. A: schematic representation of the tip part of the newly devised push pull cannula. The cut ends of two 27-gauge stainless-steel needles were sharpened and bent, then glued in such a manner that the cut ends faced each other. The tip of the glued needles had an open space at which the perfusate made contact with the tissue. The cannula was inserted into the dorsal horn to a depth of 0.7 m m from the surface at the level of L5 of the rat and the tissue was perfused at a rate o f 30/d/rain. B: effects o f noxious pinch and the forced movements of ankle joint on the release of iSP from the lumbar dorsal horn in control rats. Noxious mechanical pinches and the joint movements were given at the dotted and striped columns, respectively. Spontaneous release was calculated by the average of the a m o u n t of iSP released in the first 3 samples from each experiment and served as the control value (100%). Values represent the mean ±S.E.M.
maintained at the lowest level (unpublished data). The rats were anesthetized with sodium pentobarbital (40 mg/kg i.p.), decerebrated at the rostral end of the thalamus (ethical) and the spinal cord was exposed by laminectomy from L2-L6 levels. The animal was immobilized with gallamine and artificially respired. A newly devised pushpull cannula consisting of a pair of 27-gauge needles (Fig. 1A) was unilaterally introduced into the superficial layer of the dorsal horn at the level of Ls, in such a manner that the longer axis of the tip of the cannula paralleled the sagittal line. Superfusion of this tissue was given at a rate of 30/zl/min with a 37°C perfusate bubbled with a mixture of 95% 02-5% CO2. The perfusate included (mM): NaCI 28.5, KC1 3.0, CaCI2 1.15, MgCI2 0.85, NaHCO3 21.0, NaHPO4 0.25, glucose 3.4, and bestatin 0.01 (a gift from Nippon Kayaku). Following preperfusion for 60 min, samples were collected at 20-min intervals, iSP in the samples was assayed by radioimmunoassay as described [8]. Noxious mechanical stimuli were given by pinching (pressure in 300 g/mm 2) the clipped skin of the hind paw ipsilateral to the perfusion site with a hemostatic forceps for 30 s, and then repeated at a rate of 1/min for 20 min. As a different type of stimulation, the ankle joint was alternately flexed and extended for 15 s each with pauses of 15 s, for 20 min. Such a stimulation was innocuous in non-inflamed control rats but produced nociceptive responses like vocalization in polyarthritic intact and conscious rats. After termination of each experiment, the location of the tip
317 P
100 --
!
',.)
'5 E
50--
0.
.u2_
control rats
polyarthritic rats
(n=8)
(n=5)
Fig. 2. The amounts of spontaneous release of iSP from the lumbar dorsal horn in control and polyarthritic rats. The spontaneous release was calculated by an average of the amount of iSP released in the lirst 3 samples from each experiment. Values represent the mean_+S.E.M. Note a significant increase in the spontaneous release of iSP in polyarthritic rats.
of the cannula was histologically verified. The average amount of iSP released in the first 3 samples (spontaneous release) from each animal was used as the control value (100%), and results were expressed as mean ± S . E . M . Statistical significance was determined by means of a paired t-test, unless otherwise mentioned. In control non-inflamed rats, noxious mechanical stimulation produced by pinching the skin of the hind paw evoked a significant increase in the release of iSP from the spinal dorsal horn (e.g. 300.5 4-41.2 fmol/fraction during the first pinching period and 79.5 4- 10.6 in the period immediately before the first stimulation period; P < 0.05; "~
n=5
P
~r
800
~
"A-
"~r
aD e3
600
0
400
v
n
200 loo
~
=-
~ 1
2
3
4
h
Joint move.
Fig. 3. Effects of the forced movements of the inflamed ankle joint on the release of iSP from the lumbar dorsal horn in polyarthritic rats. The stimulations were given at the striped columns. Data processing was described in the legend for Fig. lB. Values represent the mean + S.E.M.
318
Fig. I B), while the forced movement of ankle joint (innocuous stimulation) did not affect the release ofiSP. As shown in Fig. 2, the amounts of spontaneously released iSP in the first 3 fractions were 79.8 __+_2.3 _ fmol/fraction in control rats (n = 8) but 106.9 + 6.5 fmol/fraction in polyarthritic rats (n= 5). The difference between these amounts was significant ( P < 0.05, unpaired t-test). In polyarthritic rats, the forced movements of the inflamed ankle joint (noxious stimulation) evoked a significant increase in the release of iSP from the spinal dorsal horn (e.g. 859.8_+205.3 fmol/fraction during the first stimulation period and 94.6_+8.1 in the period immediately before the first stimulating period; P<0.05). Similar increases in the release of iSP were produced in the second and third stimulation periods at intervals of 40 min (Fig. 3). The present study provides the first evidence that the spontaneous release of iSP from the spinal dorsal horn in polyarthritic rats exceeds that in control rats. The forced movements of the ankle joint, which were innocuous and did not affect the release of iSP in non-inflamed control rats, were converted to noxious stimuli and produced a significant increase in the iSP release, in polyarthritic rats. In control rats, noxious mechanical stimuli produced an increase in the iSP release, similar to that noted in the experiments using rabbits [8]. These results suggest that iSP in the perfusate includes that released from the primary afferent terminals. Electrophysiological approaches demonstrated the increased spontaneous activities and responsiveness to various peripheral stimuli of spinal dorsal horn neurons and primary sensory afterents in chronic polyarthritic rats [3, 7]. These findings suggest that the facilitated responsiveness of the spinal dorsal horn neurons in arthritic animals is partly attributed to the increased release of iSP from the excessively activated primary sensory neurons. In conclusion, as the release of iSP from the spinal dorsal horn was facilitated in polyarthriti¢ rats, SP probably plays an important role in transmission of the chronic pain of inflamed joints. We thank M. Ohara for comments on the manuscript. This work was partly supported by Grant-in-aid for Scientific Research (148044) and Grant-in-aid for Special Project Research of Selected Intractable Neurological Disorders from the Ministry of Education, Sciences and Culture of Japan, and the Naito Foundation Subsidy for Promotion of Specific Research Projects. I Colpaert, F.C., Witte, P.D., Maloni, A.N., Awouters, F., Niemegeers, C.J.E. and Janssen, P.A.J., Selfadministration of the analgesic suprofen in arthritic rats: evidence of Mycobaeterium butyri~uminduced arthritis as an experimental model of chronic pain, Life Sci., 27 (1980) 921-928. 2 Colpaert, F.C., Meert, T., WiUe, P.D. and Schmitt, P., Further evidence validating adjuvant arthritis as an experimental model of chronic pain in the rat, Life Sci., 31 (1982) 67 75. 3 Guilbaud, G., Iggo, A. and Tegner, R., Sensory receptors in ankle joint capsules of normal and arthritic rats, Exp. Brain Res, 58 (1985) 29-40. 4 Henry, J.L., Effects of substance P on functionally identified units in cat spinal cord, Brain Res., 114 (1976) 439.-451. 5 H6kfelt, T., Kellerth, J.O., Nilsson, G. and Pernow, B., Experimental immunohistoehemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Res., 100 (1975) 235 252.
319
6 H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131 136. 7 Iggo, A., Guilbaud, G. and Tegner~ R., Sensory mechanisms in arthritic rat joints, in L. Kruger and J.C. Liebeskind (Eds.) Advances in Pain Research and Therapy, Vol. 6, Raven Press, New York, 1984, pp. 83 93. 8 Kuraishi, Y., Hirota, N., Sato, Y., Hino, Y., Satoh, M. and Takagi, H., Evidence that substance P and somatostatin transmit separate information related to pain in the spinal dorsal horn. Brain Res.. 325 (1985) 294 298. 9 Pircio, A.W.. Fedele, C.T. and Bierwagen. M.E., A new method for the evaluation of analgesic activity using adjuvant induced arthritis in the rat, Eur. J. Pharmacol., 31 (1975) 207 215. I0 Randic. M. and Miletic, V.~ Effect of substance P in cat dorsal horn neurons actiwLtcd by noxious stimuli, Brain Res., 128 (1977) 164 169. I I Seybold, V.S., Hylden, J.L.K. and Wilcox, G.L., Intrathecal substance P and somatostatin in rats: behaviors indicative of sensation, Peptides, 3 (1982) 49 54.
315
NSL 04456
Release of substance P from the spinal dorsal horn is enhanced in polyarthritic rats Ryoya Oku, Masamichi Satoh and Hiroshi Takagi Department ~[ Pharmacology, Faculty ( f Pharmaceutical Sciences, Kyoto University, Kvoto ~Japan) (Received 14 October 1986; Revised version received 7 November 1986: Accepted 10 November 1986} Key wordsv Substance P: Spinal dorsal horn; Release; Chronic pain; Polyarthritis; Rat Using in situ spinal dorsal horn perfusing method in decerebrated rats, we measured the release oF immunoreactive substance P (iSP) in polyarthritic rats. The spontaneous release of iSP from the dorsal horn in the polyarthritic rats was significantlyaccelerated over that in control non-inflamed rats. Passive movements of the inflamed ankle joint evoked a significant increase in the release of iSP, while a similar movement of the non-inflamedjoint led to no such increase. These results suggest that the facilitated release of iSP from the primary afferent terminals in the spinal dorsal horn in polyarthrific rats possibly plays a role in transmission of chronic pain of inflamedjoints.
Substance P (SP) is present in small diameter p r i m a r y sensory n e u r o n s [6] ternaihating in thc outer l a m i n a e of the spinal dorsal horn [5]. l o n t o p h o r e t i c a l l y applied SP has an excitatory action on some dorsal horn n e u r o n s that tire also excited by noxious stimuli [4, 10]. Moreover, intrathecal injection of the peptide elicits aversive b e h a v i o r in l a b o r a t o r y a n i m a l s [1 1]. Kuraishi et al. [8] used an in situ perfusion technique and ff)und a m a r k e d increase in release of i m m u n o r e a c t i v e SP (iSP) from the rabbit spinal dorsal horn d u r i n g acute noxious mechanical stimuli. These findings suggested that SP in the p r i m a r y sensory afferent participates in acute mechanical nociception. However, little is k n o w n of the i n v o l v e m e n t of the SP n e u r o n s in the spinal dorsal horn in chronic nociception. Thus, we directly cxamined a change in the release of" iSP from the l u m b a r dorsal horn d u r i n g chronic pain using polyarthritic rats as a reasonable model of chronic pain [I, 2, 9]. Polyarthritis was c o n v e n t i o n a l l y p r o d u c e d in male Sprague Dawley rats (220-280 g) by i n o c u l a t i n g a 0.08 ml s u s p e n s i o n of heat-killed Mycohacterium tuberculosis H 3 7 R A in paraffin oil (10 mg/ml) i n t r a d e r m a l l y into the footpad of the left hind paw. Two to 3 weeks later, the polyarthritic rats with severe swelling (foot volume, > 5.5 cm3; that of control rat is a b o u t 2.1 cm 3) were selected a n d used in the following studies. D u r i n g this period, the nociceptive thresholds in the hind paw pressure test were Correspondence." M. Satoh, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan. 0304-3940,'87:$ 03.50 @ 1987 ElsevierScientificPublishers Ireland Ltd.
316
A
B PUSH
P<0.05 vs. pre-stim release
n=3
PUU. 400
02
01
•
,
27G needle
~r
'5
space
200
~ 1oo i
0.8~ 1
~ 4
Pinch
or~)
3 Joint move.
4
Pinch
Fig. I. A: schematic representation of the tip part of the newly devised push pull cannula. The cut ends of two 27-gauge stainless-steel needles were sharpened and bent, then glued in such a manner that the cut ends faced each other. The tip of the glued needles had an open space at which the perfusate made contact with the tissue. The cannula was inserted into the dorsal horn to a depth of 0.7 m m from the surface at the level of L5 of the rat and the tissue was perfused at a rate o f 30/d/rain. B: effects o f noxious pinch and the forced movements of ankle joint on the release of iSP from the lumbar dorsal horn in control rats. Noxious mechanical pinches and the joint movements were given at the dotted and striped columns, respectively. Spontaneous release was calculated by the average of the a m o u n t of iSP released in the first 3 samples from each experiment and served as the control value (100%). Values represent the mean ±S.E.M.
maintained at the lowest level (unpublished data). The rats were anesthetized with sodium pentobarbital (40 mg/kg i.p.), decerebrated at the rostral end of the thalamus (ethical) and the spinal cord was exposed by laminectomy from L2-L6 levels. The animal was immobilized with gallamine and artificially respired. A newly devised pushpull cannula consisting of a pair of 27-gauge needles (Fig. 1A) was unilaterally introduced into the superficial layer of the dorsal horn at the level of Ls, in such a manner that the longer axis of the tip of the cannula paralleled the sagittal line. Superfusion of this tissue was given at a rate of 30/zl/min with a 37°C perfusate bubbled with a mixture of 95% 02-5% CO2. The perfusate included (mM): NaCI 28.5, KC1 3.0, CaCI2 1.15, MgCI2 0.85, NaHCO3 21.0, NaHPO4 0.25, glucose 3.4, and bestatin 0.01 (a gift from Nippon Kayaku). Following preperfusion for 60 min, samples were collected at 20-min intervals, iSP in the samples was assayed by radioimmunoassay as described [8]. Noxious mechanical stimuli were given by pinching (pressure in 300 g/mm 2) the clipped skin of the hind paw ipsilateral to the perfusion site with a hemostatic forceps for 30 s, and then repeated at a rate of 1/min for 20 min. As a different type of stimulation, the ankle joint was alternately flexed and extended for 15 s each with pauses of 15 s, for 20 min. Such a stimulation was innocuous in non-inflamed control rats but produced nociceptive responses like vocalization in polyarthritic intact and conscious rats. After termination of each experiment, the location of the tip
317 P
100 --
!
',.)
'5 E
50--
0.
.u2_
control rats
polyarthritic rats
(n=8)
(n=5)
Fig. 2. The amounts of spontaneous release of iSP from the lumbar dorsal horn in control and polyarthritic rats. The spontaneous release was calculated by an average of the amount of iSP released in the lirst 3 samples from each experiment. Values represent the mean_+S.E.M. Note a significant increase in the spontaneous release of iSP in polyarthritic rats.
of the cannula was histologically verified. The average amount of iSP released in the first 3 samples (spontaneous release) from each animal was used as the control value (100%), and results were expressed as mean ± S . E . M . Statistical significance was determined by means of a paired t-test, unless otherwise mentioned. In control non-inflamed rats, noxious mechanical stimulation produced by pinching the skin of the hind paw evoked a significant increase in the release of iSP from the spinal dorsal horn (e.g. 300.5 4-41.2 fmol/fraction during the first pinching period and 79.5 4- 10.6 in the period immediately before the first stimulation period; P < 0.05; "~
n=5
P
~r
800
~
"A-
"~r
aD e3
600
0
400
v
n
200 loo
~
=-
~ 1
2
3
4
h
Joint move.
Fig. 3. Effects of the forced movements of the inflamed ankle joint on the release of iSP from the lumbar dorsal horn in polyarthritic rats. The stimulations were given at the striped columns. Data processing was described in the legend for Fig. lB. Values represent the mean + S.E.M.
318
Fig. I B), while the forced movement of ankle joint (innocuous stimulation) did not affect the release ofiSP. As shown in Fig. 2, the amounts of spontaneously released iSP in the first 3 fractions were 79.8 __+_2.3 _ fmol/fraction in control rats (n = 8) but 106.9 + 6.5 fmol/fraction in polyarthritic rats (n= 5). The difference between these amounts was significant ( P < 0.05, unpaired t-test). In polyarthritic rats, the forced movements of the inflamed ankle joint (noxious stimulation) evoked a significant increase in the release of iSP from the spinal dorsal horn (e.g. 859.8_+205.3 fmol/fraction during the first stimulation period and 94.6_+8.1 in the period immediately before the first stimulating period; P<0.05). Similar increases in the release of iSP were produced in the second and third stimulation periods at intervals of 40 min (Fig. 3). The present study provides the first evidence that the spontaneous release of iSP from the spinal dorsal horn in polyarthritic rats exceeds that in control rats. The forced movements of the ankle joint, which were innocuous and did not affect the release of iSP in non-inflamed control rats, were converted to noxious stimuli and produced a significant increase in the iSP release, in polyarthritic rats. In control rats, noxious mechanical stimuli produced an increase in the iSP release, similar to that noted in the experiments using rabbits [8]. These results suggest that iSP in the perfusate includes that released from the primary afferent terminals. Electrophysiological approaches demonstrated the increased spontaneous activities and responsiveness to various peripheral stimuli of spinal dorsal horn neurons and primary sensory afterents in chronic polyarthritic rats [3, 7]. These findings suggest that the facilitated responsiveness of the spinal dorsal horn neurons in arthritic animals is partly attributed to the increased release of iSP from the excessively activated primary sensory neurons. In conclusion, as the release of iSP from the spinal dorsal horn was facilitated in polyarthriti¢ rats, SP probably plays an important role in transmission of the chronic pain of inflamed joints. We thank M. Ohara for comments on the manuscript. This work was partly supported by Grant-in-aid for Scientific Research (148044) and Grant-in-aid for Special Project Research of Selected Intractable Neurological Disorders from the Ministry of Education, Sciences and Culture of Japan, and the Naito Foundation Subsidy for Promotion of Specific Research Projects. I Colpaert, F.C., Witte, P.D., Maloni, A.N., Awouters, F., Niemegeers, C.J.E. and Janssen, P.A.J., Selfadministration of the analgesic suprofen in arthritic rats: evidence of Mycobaeterium butyri~uminduced arthritis as an experimental model of chronic pain, Life Sci., 27 (1980) 921-928. 2 Colpaert, F.C., Meert, T., WiUe, P.D. and Schmitt, P., Further evidence validating adjuvant arthritis as an experimental model of chronic pain in the rat, Life Sci., 31 (1982) 67 75. 3 Guilbaud, G., Iggo, A. and Tegner, R., Sensory receptors in ankle joint capsules of normal and arthritic rats, Exp. Brain Res, 58 (1985) 29-40. 4 Henry, J.L., Effects of substance P on functionally identified units in cat spinal cord, Brain Res., 114 (1976) 439.-451. 5 H6kfelt, T., Kellerth, J.O., Nilsson, G. and Pernow, B., Experimental immunohistoehemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Res., 100 (1975) 235 252.
319
6 H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131 136. 7 Iggo, A., Guilbaud, G. and Tegner~ R., Sensory mechanisms in arthritic rat joints, in L. Kruger and J.C. Liebeskind (Eds.) Advances in Pain Research and Therapy, Vol. 6, Raven Press, New York, 1984, pp. 83 93. 8 Kuraishi, Y., Hirota, N., Sato, Y., Hino, Y., Satoh, M. and Takagi, H., Evidence that substance P and somatostatin transmit separate information related to pain in the spinal dorsal horn. Brain Res.. 325 (1985) 294 298. 9 Pircio, A.W.. Fedele, C.T. and Bierwagen. M.E., A new method for the evaluation of analgesic activity using adjuvant induced arthritis in the rat, Eur. J. Pharmacol., 31 (1975) 207 215. I0 Randic. M. and Miletic, V.~ Effect of substance P in cat dorsal horn neurons actiwLtcd by noxious stimuli, Brain Res., 128 (1977) 164 169. I I Seybold, V.S., Hylden, J.L.K. and Wilcox, G.L., Intrathecal substance P and somatostatin in rats: behaviors indicative of sensation, Peptides, 3 (1982) 49 54.