Brain Research, 568 (1991) 109-115 ~) 1991 Elsevier Science Publishers B.V. All fights reserved. 0006-8993/911503.50
109
BRES 17281
Release of substance P into the superficial dorsal horn following nociceptive activation of the hindpaw of the rat Kenneth E. McCarson and Barry D. Goldstein Department of Pharmacology and Tomcology, Medical College of Georgm, Augusta, GA 30912 (U.S.A.) (Accepted 6 August 1991)
Key words: Substance P; Release; Dorsal horn; Formalin test; Nociception; Rat
Substance P (SP) has been widely proposed as being involved in the transmission of nociceptive information in the dorsal horn of the spinal cord. Formalin rejected into the hindpaw as a nociceptive sumulus has been shown to increase the amount of immunoreactive SP in the dorsal horn, perhaps by decreasing SP release from primary afferent neurons. Much is known concerning the release of SP from tissue slices or from the entire spinal cord m vivo. However, less is known about the release patterns of SP in the superficial dorsal horn during the activation of peripheral nociceptors. In this study, noxious pinch applied to and formalin injection into the hindpaw were used as nociceptive stimuli while a stereotaxic push-pull eannula was used to perfuse the L5 dorsal horn. Experiments were conducted in unanesthetized decerebrate/spinal rats, and radioimmunoassay was used to determine the SP-hke lmmunoreactivity (SPLI) content of collected perfusates. Results demonstrate that graded intensities of noxious mechamcal pinch produced progressively increased release of SPLI into the dorsal horn; SPLI release returned to baseline rates following termination of the stimulus. The injection of 100/~1 of 5% formalin into the hindpaw produced a biphasic inhibition of SPLI release 0-40 rain and >60 min after formalin injection. The application of a noxious pinch following formalin rejection produced an increase in SPLI release which did not return to baseline rates; this may be indicative of production of a hyperalgesic state caused by formalin injection. The results of this study support the concept that formalin injected into the lundpaw activates segmental antinociceptive systems which block SP release and hmlt nociceptive transmission. These results support evidence that SP may play a limited role in the response to formalin. INTRODUCTION The undecapeptide substance P (SP) is contained in the central terminals of small diameter primary afferent neurons which terminate in the superficial laminae of the dorsal horn of the spinal cord 19. Dorsal rhizotomy decreases dorsal horn SP content by 80%; the other 20% is contained in intrinsic or descending neurons 26. The A~ and C fibers containing SP can be activated by a number of noxious stimuli and respond by releasing SP into the dorsal horn 5'11'15'27-29'35'36'42. The actions of SP in the spinal cord are widely accepted as being involved in the transmission of nociceptive information 17'3s. Iontophoretic application of SP onto dorsal horn neurons has been shown to excite the same neurons as noxious peripheral stimulation 16. Furthermore, there is a large body of evidence associating intrathecal injections of SP with nociceptive behavior 24'39 and decreased nociceptive threshold 25, although there is some evidence to the contrary2,3,13,14. The formalin test has been widely accepted as a model of chemogenic pain 1°'22'41. The injection of dilute formalin into the paw of animals has been shown to produce a
biphasic response with both early and late phases of pain-related behavior 1°'22'41. Electrophysiological studies have demonstrated similar biphasic increases in the excitability of dorsal horn cells following formalin injection into their receptive fields 8'9. It has also been shown that the injection of formalin into the hindpaw as a nociceptive stimulus produces a biphasic increase in dorsal horn SP levels 32. These increases in dorsal horn SP-like immunoreactivity (SPLI) may be due to decreased SP release from primary afferent neurons 32. There is evidence that endogenous opiate systems are activated by the injection of formalin into the hindpaw 4'31'32. Both SP levels and release may be regulated by the primary afferent neuron during nociception. Much is known concerning the release of SP from tissue slices 3°' 36,37 or from the entire spinal cord in vivo 15'42. However, less is known about the release patterns of SP at specific sites in the terminal fields of small diameter primary afferent neurons when their specific receptive fields are activated. Satoh and his coworkers have performed several studies demonstrating the release of SP into the dorsal horn in both decerebrate/anesthetized rabbits and rats 27-29'35. This study was undertaken to determine the
Correspondence. B.D. Goldstein, Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300, U.S.A.
110
tern of S P L I in the superficial dorsal h o r n o f the l u m b a r
mg outflow was 2 8 - 0.26 pg SPLU100/zl. This corresponded to a recovery of 56% of the SP concentration in the standard solution.
spinal c o r d during the application o f a c u t e and s u b a c u t e
Radtotmmunoassay
n o c i c e p t i v e stimuli to the h i n d p a w s of rats.
The primary antisera was procured from Peninsula laboratories and supplied as lyophilized powder. A dilution was chosen that yielded approximately 45% specific binding of the 15,000 cpm of tracer added to each assay tube. The tracer used was 123I-[Tyr8] substance P as supphed by New England Nuclear. The assay buffer was a phosphate-buffered saline (pH 7.4) having the following composition: Na2HPO 4 9 raM, NaH2PO 4 41 raM, NaC1 0.14 M, bovine serum albumin 0.75%, and NaN 3 0.02%. Standards, blanks, and total binding tubes each received 200 #l of CSF m order to match unknown samples. Each tube was vortexed and allowed to incubate for at least 24 h at 4°C. An activated charcoai/dextran suspension was used to separate the free tracer. The resulting standard curve typically had an ICso of about 15 pg SP/assay tube and minimum detectable limit of about 1 pg SP/assay tube The primary antlsera cross-reacts with other SP related peptldes as follows: SP(5-11), 30%; SP-(3-11), 19%; SP-(7-11), 14%; SP-(1-4), less than 2.5%; and SP-(1-6), SP-(1-7), SP-(1-9), eledoisin, neurokmm A, neurokmin B, SP-GIy (fl-preprotachylonin)-(58-69)), and SP-GlyLys (fl-preprotachykinin-(58-70)) all less than 1% (all values calculated on a percentage molar basis).
effects o f n o c i c e p t o r activation o n the local release pat-
MATERIALS AND METHODS
Dorsal horn perfusion SPLI release was studied using a mochfication of a method described by Oku et al. as. Briefly, rats were anesthetized with halothane, immohilized with succinylcholine, and artificially respired. A dorsal laminectomy was performed to expose the lumbar enlargement The lammectomy was extended rostrally about 1 cm to provide a space where an acute spinal transection was performed. A carotid arterial cannula was inserted through which blood pressure was momtored throughout the experiment. The rat was then decerebrated at the level of the collicull and the anesthetic removed Following surgery, the rat was placed in a spinal unit and body temperature maintained with a thermistor-regulated heating pad placed under the animal. The pia mater was opened and a push-pull cannula devised from two 27-gauge needles was stereotaxacally introduced into the dorsal horn at the entry zone of the fifth lumbar dorsal root, 1 mm from the midline and approximately 700 #m deep. In each animal only one perfusion site was established. A peristaltic pump was used to introduce and remove artlficaal cerebrospinal fluid (CSF) through the cannula at a rate of 30 #l/min. The artificial CSF contained (in raM): NaC1 128.5, KCI 3.0, NaHCO a 21.0, NaHzPO 4 0.25, glucose 3.4, CaCI2 1.15, MgC12 0.8 and (in #M): bestatin 10, captopril 5, leupeptin 1.0, and chymostatin 1.0. The CSF was bubbled with 95% 02/5% CO2 to maintmn a pH of 7.4 and warmed to 37°(2 immediately before entering the cannula. Perfusion continued for at least 1 h before collection of samples for radiolmmunoassay (RIA) for SP. Perfusates were collected d~rectly into ~ce-cold assay tubes which contmned SP ant~sera. Samples (200/d) were collected into each assay tube every 6.66 rain. Following termination of the experiment, the location of the cannula tip in the dorsal horn was verified histologically. Animals m which the perfuston area was outside of laminae I and II of the dorsal horn or with cardiovascular events which altered SPLI release (such as blood pressure spikes or drops to less than 50 mm Hg) were excluded from the study.
Experimental protocol For each experiment, SPLI release was momtored for 60 mm prior to hindpaw treatment to establish a baseline control release rate for each ammal. The rats then received a 100 #1 injection of either 5% formalin or sterile isotonic saline into the plantar aspect of the hindpaw, or one of 3 intensities of mechanical pinch. Low and medium intensRy pinches were applied using small bulldog clamps with jaw opening weights of 50 and 250 g, respectively. Each of these pinches was delivered for 30 s each minute for 20 min to various loeatmns on the hindpaw and lower limb. A high intensity pinch was applied using a hemostatlc forceps w~th a jaw opening weight of approximately 1000 g applied once to the hindpaw for the entire 20-rain stimulus period. Jaw opening weights were measured by immoblhzmg one jaw of the forceps and hangmg weights from the lower jaw until the weight necessary to just open the forceps was reached. All stimuli were applied to the hindpaw lpsilateral to the site of dorsal horn perfuslon. Each expenmental subject received only one stimulus, with the exception of one group of animals in which the medmm intensity pinch was applied 90 min after formalin injeetaon. An experiment to determine the recovery of SP by the push-pull cannula was performed m which the cannula was immersed in a CSF solution containing SP and CSF moved through the cannula at the same rate as in animal experiments. The resulting cannula effluent was then assayed for SP content. When the push-pull cannula was inserted m a CSF containing 5 0 pg SP/100/zl, the result-
Data analysts SPLI release data were collected as pg SPLI/sample; m the figures, release data are shown as percent of basehne release and were compared to the sample period immediately preceding stimulus application. Thus, in the inset data are normalized to the 6.66-min period preceding hindpaw treatment; m the larger graph, data are normalized to the 20-min period preceding hmdpaw treatment. This longer baseline penod includes additional data not used in the reset Although this results in graphs which appear different, the raw data are identical for the treatment periods of each figure. Both graphs are included in order to show the nature of the raw data as collected (6.66-rmn intervals) and to facilitate the analysis of general trends in the data (20-min intervals). Analysis of covanance (ANCOVA) was used to test the raw data for statistical significance. Tukey's HSD test was used to perform post-hoc comparisons. Significance was set at P < 0.05, and all values are reported as the mean - S E.M
RESULTS Table I shows the n u m b e r of subjects u s e d for e a c h t r e a t m e n t and w h e t h e r t h e i r g e n e r a l t r e n d was t o w a r d i n c r e a s e d o r d e c r e a s e d S P L I r e l e a s e rates. T h e baseline S P L I release rates v a r i e d widely b e t w e e n e x p e r i m e n t a l subjects. T h e m e a n b a s e l i n e r e l e a s e r a t e was 6.63 --- 0.96 pg S P L I / s a m p l e (0.99 pg S P L I p e r m i n u t e ) . T h e variation of the 60-min b a s e l i n e p e r i o d f r o m s a m p l e to sample (6.66 m i n ) a v e r a g e d 12% of the b a s e l i n e that was used for the c o m p a r i s o n o f t h e n o c i c e p t i v e stimulus. Fig. 1 shows the r e l e a s e p a t t e r n of S P L I f o l l o w i n g application o f t h e low intensity pinch to the h i n d p a w . This pinch was mildly n o x i o u s w h e n a p p l i e d to t h e w e b b i n g b e t w e e n the i n v e s t i g a t o r ' s fingers. It can be s e e n in Table I that this pinch p r o d u c e d i n c r e a s e d S P L I release in half of t h e recipients. In the rat it p r o d u c e d a 17% increase in the a m o u n t of S P L I r e l e a s e d into the ipsilateral L 5 dorsal h o r n during the 20 m i n of stimulus application (Fig. 1). W h e n v i e w e d in 6.66-min samples (see
111 TABLE I Changes in SPLI release in individual subjects Treatment
n
Low intensity pinch 6 Medium intensity pinch 13 High intensity pinch 3 Formahn 6 Saline 7 Medium mtenslty pinch after formahn 4
Number (%) of ammals showing:* Increase
Decrease No change
3 (50%) 12 (92%) 3 (100%) 0 0
0 1 (8%) 0 6 (100%) 7 (100%)
4 (100%) 0
3 (50%) 0 0 0 0 0
* Mean of SPLI release values during treatment (20 min for pinches, 80 min for formalin and saline) compared to mean of 20 min baseline for same experimental subject. inset), it can be seen that SPLI release was increased by as much as 110%. This increase was significantly different from baseline release only when the d a t a were l u m p e d into 20-min periods. T h e differences in a p p e a r ance of the d a t a shown in each figure and its inset are due to the calculation of percent of control values with respect to different periods of baseline release values (see Materials and Methods). Fig. 2 shows the release p a t t e r n of SPLI into the dorsal horn following application of the m e d i u m intensity pinch to the hindpaw. This pinch was frankly noxious
A lOO
B 2O0
when applied to the investigator, and Table I shows that it p r o d u c e d increased SPLI release in 92% of recipients. It p r o d u c e d an increase in SPLI release which was greater in both intensity and duration than the low intensity pinch (Fig. 2). W h e n viewed in 20-min periods, SPLI release increased to 33% of baseline rates during the application of the stimulus, r e m a i n e d elevated (26%) during the 20 min following termination of the stimulus, and r e t u r n e d to baseline 20 min after termination of the stimulus. The inset shows that when viewed in 6.66-rain periods, similar increases were seen (25-30%) during the application of the stimulus, but SPLI release was not significantly different from baseline release rates as soon as the stimulus was terminated. The effects of the high intensity pinch are shown in Fig. 3. This pinch p r o d u c e d inflammation and tissue injury in the hindpaw of the rat and was not tested by application to the investigator. Table I shows that all animals receiving the high intensity pinch r e s p o n d e d with increased SPLI release. It p r o d u c e d a 146% increase in SPLI release during the 20-min stimulus period; when viewed in 6.66-rain periods the increase was as great as 587% (Fig. 3). This increase was significant c o m p a r e d to both 6.66- and 20-min baseline periods; however, the increase was short-lived c o m p a r e d to the m e d i u m intensity pinch. L o o k i n g at the inset, it can b e seen that release r e t u r n e d to slightly below baseline immediately following termination of the pinch. The time course of SPLI release following formalin injection into the hindpaw is shown in Fig. 4. F o r m a l i n p r o d u c e d a significant decrease in the release of SPLI.
I
L
0
~-g
i
h
20
1 Low
Intensity
40 Pinch
50
A
B
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..=
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, 60
~ 50 E O
o -20 Low IntensntyPinch
i i i 0 20 40 I Medium Intenstty Pinch
-50
0 T
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20 40 Time (minutes)
Fig. 1. Effects of low intensity pmch on SPLI release. Graphs show the time course of SPLI release in the dorsal horn of animals receiving a low intensity noxious pinch applied to the hindpaw (n = 6). The pinch was applied during the time period denoted by the cross-hatched area of the graph. B shows data resulting from 6.6re.in sample periods and percentages of control are compared to the one 6.6-rain sample period immediately preceding stimulus application. In A, each bar represents the same data lumped into 20rain sample periods for clarity; percents of control are then based on the mean of the three 6.6-rain sample periods immediately preceding application of the stimulus. *P < 0.05 as compared to baseline release (analysis performed on raw data).
I g
o
-20 Medium Intensity Pinch
I
0 T
20
40 60 Time (minutes)
Fig. 2. Effects of medium intensity pinch on SPLI release. Graphs show the time course of SPLI release in the dorsal horn of animals receiving a medium intensity noxious pinch applied to the hindpaw (n = 13). The pinch was applied during the time period denoted by the cross-hatched area of the graph. Data are reported as in Fig. 1.
112
B
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B 1000
0 ~
I
5
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_= 50 •" 400
0 20 l H i g h Intensity P m c h
-100 [
40
0
20 rSahne
40
60
80
©
gE
o
0
e1
~ -50
o
-100
-20 H~gh Intensity Pinch
0 T
.... -20
20 40 Ttme (minutes)
20
40
Sahne
F~g. 3. Effects of high-intensity pinch on SPLI release. Graphs show the time course of SPLI release in the dorsal horn of animals receiving a high intenmy noxious pinch applied to the hindpaw (n = 3). The pinch was apphed during the time period denoted by the cross-hatched area of the graph. Data are reported as m Fig. 1.
It can be seen in the inset that formalin produced a biphasic effect, with SPLI release being inhibited by up to 60% for the first 40 min after injection into the hindpaw and again after 60 rain following formalin injection. After 80 min SPLI release remained suppressed in experiments which were not terminated by that time (data not shown for clarity). The effects of the control injection of saline are shown in Fig. 5. Although there was a trend toward saline decreasing SPLI release, none of the effects were significantly different from baseline release rates when analyzed in 6.66- or 20-rain intervals. Table I
60 80 Time (minutes)
Fig. 5. Effects of saline on SPLI release. Graphs show the ume course of SPLI release in the dorsal horn of ammals recelvang an rejection of 100/~! of sahne into the hindpaw (n = 7). Data are reported as in Fig. 1.
shows that both saline and formalin produced decreased SPLI release in all subjects. Fig. 6 shows the effect of the medium intensity pinch when applied approximately 90 min following formalin injection into the hindpaw. It can be seen that during application of the pinch SPLI release was increased to a degree similar to that in normal animals in both 20- and 6.66-min periods (see Fig. 2). However, in the formalin injected animals SPLI release did not return to baseline rates; instead, SPLI release progressively increased to as
B
A I000
AI®
1ooo ;
-
I
-50
~" e~
0
50
100
I) r
- -
0 20 40 60 T M e d i u m lntenstty Pinch After F o r m a h n
500 t 0 20 l Formahn
40
60
80
O
E
~
-50
T -20 0 Medtum IntensRy, Pinch t Atter rormaun /
-100
-20
0 20 T Fonnahn
40
60 8(1 Time (minutes)
Fig. 4 Effects of formahn on SPLI release. Graphs show the time course of SPLI release in the dorsal horn of ammals recewmg an rejection of 100/~1 of formahn into the hindpaw (n = 6). Data are reported as m Ftg 1
20
40 60 Time (minutes)
Fig. 6. Effects of medium intensity pinch after formahn on SPLI release. Graphs show the time course of SPLI release in the dorsal horn of animals recewmg a medium intensity noxious pinch applied to the hmdpaw 90 mm after injection of 100/A of formalin into the same lundpaw (n = 4). The pinch was applied during the time pen o d denoted by the cross-hatched area of the graph. Data are reported as in Fig. 1
113 much as 389% (20-min period) or 1400% (6.66-min period) following termination of the stimulus. These increases were only significant when analyzed in 20-min periods. Table I shows that application of the medium intensity pinch after formalin uniformly produced increases in SPLI release. DISCUSSION Much information has been gained concerning the release of SP in the spinal cord using techniques such as perfusion of tissue slices3°'36'37 or the entire spinal cord 15' 42. Such methods are limited in their ability to localize the specific site of SP release. Antibody-coated microprobes 11 and microdialysis probes 5 have been successfully used to localize SP release but may be limited in their ability to quantify the amount of peptide released 7. The stereotaxic push-pull cannula used in this study and studies by other investigators 27-29'35 can localize not only the specific site but also the quantity of released neurotransmitters. There is evidence that push-pull perfusion techniques may yield much higher recovery of peptides than microdialysis techniques 7. The recovery of 56% of a perfused solution in this study demonstrates the ability of the push-pull cannula to assess peptide content with a degree of accuracy which may not be possible with techniques which rely on diffusion of peptides through a dialysis membrane. Here we report the release patterns of SPLI into the L5 superficial dorsal horn following the application of various nociceptive stimuli to the hindpaw of unanesthetized decerebrate/spinal rats. It is likely that the majority of the SPLI released into the dorsal horn is actual substance P. N-terminal SP fragments are found in rat CNS 4°, and may have biological activity6, but show very little crossreactivity toward the SP antisera used in this study. The primary antisera only cross reacts, to any appreciable degree, with C-terminal SP fragments, primarily SP-(5-11) (30%). The vast majority of SP C-terminal fragments found in the rat CNS coelute with SP-(511) in gel electrophoresis4°, and a metabolically protected SP-(5-11) analogue 12 has been shown to have biological activity in rat CNS. However, it has been shown that pyroglutamicS-SP-(5-11) (the naturally occurring cyclized metabolite of SP-(5-11) is not present in the potassium stimulated release products of isolated rat spinal cord 1. Thus, SP-(5-11) probably does not contribute to the SPLI measured in these studies. Primary afferent neurons which contain SP are probably the major source of the SPLI release measured in this study. Most of the SP content of the dorsal horn is contained in primary afferent terminals 26. The superficial dorsal horn areas perfused in this study correspond
with the terminal fields of small diameter primary afferent neurons which contain SP 19. The contribution of evoked SPLI release from descending neurons is unlikely in the spinal transected animals used in this study. Furthermore, other investigators have shown that the SP content of dorsal horn tissues remains unchanged when intrinsic neurons are destroyed by the occlussion of the spinal cord blood supply21. The increased SPLI release seen during noxious mechanical stimulation is consistent with results of other studies 15'29. This study also demonstrates a graded response of increasing SPLI release with increased intensity of the pinch applied to the hindpaw. There is a distinct increase in the magnitude and duration of the evoked SPLI release between the low and medium intensity noxious pinches; this is consistent with the activation of nociceptive afferent neurons. The high intensity pinch showed a further increase in the amount of SPLI released, but the duration of the response was attenuated. This intense stimulus produced inflammation and tissue damage which may have activated systems which respond to limit SPLI release, an effect similar to that produced by formalin. Alternatively, the high intensity pinch may have simply depleted the terminal content of SP available for release into the dorsal horn. In this study, formalin clearly produced a decrease in the amount of SPLI released into the dorsal horn. The physiology of the changes induced by formalin is poorly understood, but there is evidence that an endogenous opiate system plays a role in the control of the transmission of nociceptive information. The intense nociceptive activation produced by formalin activates opiate antinociceptive systems which act to produce a buildup of SPLI content in the dorsal horn 31'32. Although the effects of descending inhibitory systems have been removed in these experiments by acute spinal transection, the existence of enkephalinergic segmental control systems activated by noxious stimuli has been demonstrated 43. The segmental release of Met-enkephalin has been shown to increase following formalin injection into the hindpaw 4. Additionally, the opiate antagonist naloxone has been shown to increase SP release 15 and potentiate the pain-related behavior evoked by noxious stimuli41. Thus, there is evidence that the dynamics of the responses of SP to nociceptive stimuli are in part under the control of a system of endogenous opioid peptides that limit the transmission of nociceptive information. This study provides evidence that these systems may act by blocking the release of SP from primary afferent neurons after an intense stimulus such as formalin injection. In this study, no increases in SPLI release were seen following formalin injection such as those described by Kuraishi et al. 29. It is, of course, possible that there is
114 an initial increase in SPLI release within the first sample collected which is of short enough duration to be masked by decreased release for the remainder of the sample period. However, we feel this is unlikely given the magnitude of the decrement in SPLI release evident in the samples immediately following formalin injection. Interestingly, saline injection into the hindpaw also tended to decrease SPLI release, but not to a significant degree. It is possible that the needle prick itself is sufficient to midly activate the antinociceptive systems which decrease SPLI release in response to formalin. Alternatively, the volume of the injection may activate lowthreshold primary afferent systems which act to limit the release of SP and the transmission of nociceptive information. Gate control systems activated by non-noxious stimuli have been proposed 33, but there is evidence that A fiber electrical stimulation does not decrease SPLI release evoked by noxious stimuli 23. It is also shown in this study that the application of the same intensity of noxious pinch to normal and formalin injected animals produces markedly different patterns of SPLI release. The progressive increases in SPLI release following termination of the pinch may reflect a hyperalgesic state of nociceptors in the hindpaw produced by the injection of formalin. Clearly, whereas SPLI release returns to baseline rates soon after the termination of noxious stimulation of normal hindpaws, SPLI release remains elevated and even increases further when the same stimulus is applied to formalin-injected hindpaws. A previous report by Kuraishi, et al. 29 has shown increases in SPLI release lasting exactly 20 rain in response to all the types of nociceptive stimuli used, including pinch, intense heat and formalin. In this study we demonstrate similar increased SPLI release due to noxious mechanical stimulation, but marked long-lasting de-
creases in the SPLI release following injection of formalin into the hindpaw. The studies conducted by Kuraishi et al. used similar methods, but were conducted using decerebrate/anesthetized rabbits with intact descending control systems and the possibility of anesthetic effects 2729. Either of these differences from the preparation used in this study could account for the differences seen in the response to formalin. Both pentobarbital and urethane anesthesia masked evoked SPLI release in our preparation (unpublished observations). We feel that the effects of formalin on release in this study may be more consistent with its long-term effects on nociceptive behavior 1°'22'41, dorsal horn neuron excitability s'9, and dorsal horn SPLI content 31'32. Finally, it must be considered that formalin may not be a purely nociceptive agent when used to activate SP containing neuronal systems. Increases in dorsal horn SPLI content stimulated by formalin can be attenuated by administration of naloxone 31'32, yet naloxone has little, if any, effect on formalin-induced nociceptive responses 18'24. Similarly, blockade of formalin-induced nociceptive behaviors by pretreatment with lidocaine does not reduce the concomitant increases in dorsal horn SPLI content 2°. Also, intrathecal injections of an SP antagonist or SP antiserum inhibit nociceptive behavior only in the early phase of the formalin response 34. Taken together with the present demonstration that SPLI release is suppressed at the same time that nociceptive behavior would be elevated, the evidence seems to suggest that SP may not be directly involved in the actual transmission of nociceptive information at least during the late phase of the formalin test.
REFERENCES
(1990) 323-329. 5 Brodin, E., Linderoth, B., Gazehus, B. and Ungerstedt, U., In vivo release of substance P in cat dorsal horn studied with mlcrodialysis, Neurosci. Lett., 76 (1987) 357-362. 6 Cridland, R.A. and Henry, J L , N- and C-terminal fragments of substance P: spinal effects in the rat tail flick test, Brain Res. Bull., 20 (1988) 429-432. 7 DeMesqmta, S., Laltmen, K., Bemfield, M., Rokaeus, A. and Crawley, J.N., Comparison of microdialysis and push-puU perfusion techniques for in vivo studies of neuropeptide release, Soc. Neurosct Abstr., 15 (1989) 213. 8 Dlckenson, A.H. and Sullivan, A.F., Peripheral ongms and central modulation of subcutaneous formalin-induced acuwty of rat dorsal horn neurones, Neurosct Lett., 83 (1987) 207-211. 9 Diekenson, A H. and Sulhvan, A.F., Subcutaneous formalinreduced activity of dorsal horn neurons m the rat: differential response to an mtrathecal opiate admimstered pre or post formalin, Pare, 30 (1987) 349-360. 10 Dubuisson, D and Dennis, S.G., The formahn test: a quantitatwe study of the analgesic effects of morphine, meperidine,
1 Akagi, H., Otsuka, M. and Yanaglsawa, M., Identification of high-performance liquid chromatography of immunoreactive substance P released from isolated rat spinal cord, Neurosct. Lett., 20 (1980) 259-263. 2 Bossut, D., Frank, H. and Mayer, D.J., Is substance P a pnmary afferent neurotransmitter for nooceptive input? II. Splnahzation does not reduce and intrathecal morphine potentiates behavioral responses to substance P, Brain Research, 455 (1988) 232-239. 3 Bossut, D., Frank, H. and Mayer, D.J., Is substance P a primary afferent neurotransmitter for noclceptwe input? IV. 2-Armno-5-phosphonovalerate (APV) and [D-pro2,D-trp7'9]substance P exert different effects on behaviors induced by mtrathecal substance P, strychnine and kainic acid, Brain Research, 455 (1988) 247-253. 4 Bourgoin, S., Le Bars, D., Clot, A.M., Hamon, M. and Cesselin, E, Subcutaneous formahn induces a segmental release of met-enkephalin-like material from the rat spinal cord, Pain, 41
Acknowledgement This study was supported in part by a grant from the Medical College of Georgia Research Institute, Inc.
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25
26
and brain stem stimulation in rats and cats, Pain, 4 (1977) 161174. Duggan, A.W., Morton, C.R., Zhao, Z.Q. and Hendry, I.A., Noxious heating of the skin releases immunoreactive substance P in the substantia gelatinosa of the cat: a study with antibody wacroprobes, Brain Research, 403 (1987) 345-349. Eison, A.S., Iverson, S.D., Sandberg, B.E.B., Watson, S.P., Hanley, M.R. and Iversen, L.L., Substance P analogue, DiMeC7: evidence for stability in rat brain and prolonged central actions, Science, 215 (1982) 188-190. Frenk, H., Bossut, D., Urca, G. and Mayer, D.J., Is substance P a pnmary afferent neurotransrmtter for nooceptlve input? I. Analysis of pain-related behaviors resulting from intrathecal administratmn of substance P and 6 excitatory compounds, Brain Research, 455 (1988) 223-231. Frenk, H., Bossut, D. and Mayer, D.J., Is substance P a primary afferent neurotransmitter for nociceptive input? III. Valprolc acid and chlordiazepoxide decrease behaviors elicited by intrathecal injection of substance P and excitatory compounds, Brain Research, 455 (1988) 240-246. Go, V.W. and Yaksh, T.L., Release of substance P from the cat spinal cord, J. Physiol., 391 (1987) 141-167. Henry, J.L., Effects of substance P on functmnaUy identified units in cat spinal cord, Brain Research, 114 (1976) 439-451. Henry, J.L., Substance P and pain: an updating, Trends Neurosci., 3 (1980) 95-97. Henry, J.L., Naloxone fails to block substance P-reduced excitation of spinal nociceptive units, Brain Res. Bull., 10 (1983) 727-730. HOkfelt, T., Kellerth, J.O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. Holland, L.N. and Goldstein, B.D., Changes in substance P-like lmmanoreactivity in the dorsal horn are associated with the 'phasic' behavioral response to a formalin stimulus, Brain Research, 537 (1990) 287-292. Homma, S., Suzulu, T., Murayama, S. and Otsuka, M., Amino acid and substance P contents in spinal cords of cats voth experimental hind-limb rigidity produced by occlusion of spinal cord blood supply, J. Neurochem., 32 (1979) 691-698. Hunskaar, S., Fasmer, O.B. and Hole, K., Formalin test in mice, a useful technique for evaluating mild analgesics, J. Neurosci. Meth., 14 (1985) 69-76. Hutchison, W.D. and Morton, C.R., Electrical stimulation of pnmary afferent A fibres does not reduce substance P release m the dorsal horn of the cat, Pain, 37 (1989) 357-363. Hylden, J.L.K. and Wilcox, G.L., Intrathecal opioids block a spinal action of substance P in mice: functional importance of both mu- and delta-receptors, Eur. J Pharmacol., 86 (1983) 95-98. Hylden, J.L.K. and Wdcox, G.L., Pharmacological characterization of substance P-induced nociceptmn in mice: modulation by opmid and noradrenergic agonists at the spinal level, J. Pharmacol. Exp. Ther, 226 (1983) 398-404. Jessell, T., Tsunoo, A., Kanazawa, I. and Otsuka, M., Substance P: depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons,
Brain Research, 168 (1979) 247-259. 27 Kuraishi, Y., Hirota, N., Sugimoto, M., Satoh, M. and Takagi, H., Effects of morphine on noxious stimuli-induced release of substance P from rabbit dorsal horn in vivo, Life Sci., Suppl. 1, 33 (1983) 693-696. 28 Kuraishi, Y., Hirota, N., Sato, Y., Kaneko, S., Satoh, M. and Takagi, H., Noradrenergic inhibition of the release of substance P from the primary afferents in the rabbit spinal dorsal horn, Brain Research, 359 (1985) 177-182. 29 Kuraishi, Y., Hirota, N., Sato, Y., Hanashima, N., Takagi, H. and Satoh, M., Stimulus specificity of peripherally evoked substance P release from the rabbit dorsal horn in sltu, Neuroscience, 30 (1989) 241-250. 30 Lembeck, E and Donnerer, J., Opioid control of the function of primary afferent substance P fibres, Eur. J Pharmacol, 114 (1985) 241-246. 31 McCarson, K.E. and Ooldstein, B.D., Naloxone blocks the formatin-induced increase of substance P in the dorsal horn, Pain, 38 (1989) 339-345. 32 McCarson, K.E. and Goldstein, B.D., Timecourse of the alteration m substance P levels in the dorsal horn following formalin: blockade by naloxone, Pare, 41 (1990) 95-100. 33 Melzack, R. and Wall, ED., Pain mechanisms: a new theory, Science, 150 (1965) 971-979. 34 Ohbuko, T., Shibata, M., Takahashl, H. and Inoki, R., Roles of substance P and somatostatin on transmission of nociceptive information induced by formalin in spinal cord, J. Pharmacol. Exp. Ther., 252 (1990) 1261-1268. 35 Oku, R., Satoh, M. and Takagi, H., Release of substance P from the spinal dorsal horn is enhanced in polyarthritic rats, Neurosci. Lett., 74 (1987) 315-319. 36 Otsuka, M. and Konishi, S., Release of substance P-like immunoreactivlty from isolated spinal cord of newborn rat, Nature, 264 (1976) 83-84. 37 Pang, I. and Vasko, M.R., Morphine and norepinephrine but not 5-hydroxytryptamine and gamma-aminobutyric acid inhibit the potassium-stimulated release of substance P from rat spinal cord slices, Brain Research, 376 (1986) 268-279. 38 Pernow, B., Substance P, Pharmacol Rev., 35 (1983) 85-141. 39 Piercey, M.E, Dobry, P.J.K., Schroeder, L.A. and Einspahr, EJ., Behavmral evidence that substance P may be a spinal cord sensory neurotransmitter, Brain Research, 210 (1981) 407-412. 40 Sakurada, T., Le Greves, P., Stewart, J. and Terenius, L., Measurement of substance P metabolites in rat CNS, J. Neurochem., 44 (1985) 718-722. 41 Sugimoto, M., Kuraishi, Y., Satoh, M. and Takagi, H., Involvement of medullary opioid-peptidergic and spinal noradrenergic systems in the regulation of formalin-induced persistent pain, Neuropharmacology, 25 (1986) 481-485. 42 Yaksh, T.L., Jessell, T.M., Gamse, R., Mudge, A.W. and Leeman, S.E., Intrathecal morphine inhibits substance P release from mammalian spinal cord in vivo, Nature, 286 (1980) 455457. 43 Yaksh, T.L. and Elde, R.E, Factors governing the release of methionine enkephahn-like immunoreactiwty from mesencephalon and spinal cord of the cat in vivo, J Neurophysiol., 46 (1981) 1056-1075.
109
BRES 17281
Release of substance P into the superficial dorsal horn following nociceptive activation of the hindpaw of the rat Kenneth E. McCarson and Barry D. Goldstein Department of Pharmacology and Tomcology, Medical College of Georgm, Augusta, GA 30912 (U.S.A.) (Accepted 6 August 1991)
Key words: Substance P; Release; Dorsal horn; Formalin test; Nociception; Rat
Substance P (SP) has been widely proposed as being involved in the transmission of nociceptive information in the dorsal horn of the spinal cord. Formalin rejected into the hindpaw as a nociceptive sumulus has been shown to increase the amount of immunoreactive SP in the dorsal horn, perhaps by decreasing SP release from primary afferent neurons. Much is known concerning the release of SP from tissue slices or from the entire spinal cord m vivo. However, less is known about the release patterns of SP in the superficial dorsal horn during the activation of peripheral nociceptors. In this study, noxious pinch applied to and formalin injection into the hindpaw were used as nociceptive stimuli while a stereotaxic push-pull eannula was used to perfuse the L5 dorsal horn. Experiments were conducted in unanesthetized decerebrate/spinal rats, and radioimmunoassay was used to determine the SP-hke lmmunoreactivity (SPLI) content of collected perfusates. Results demonstrate that graded intensities of noxious mechamcal pinch produced progressively increased release of SPLI into the dorsal horn; SPLI release returned to baseline rates following termination of the stimulus. The injection of 100/~1 of 5% formalin into the hindpaw produced a biphasic inhibition of SPLI release 0-40 rain and >60 min after formalin injection. The application of a noxious pinch following formalin rejection produced an increase in SPLI release which did not return to baseline rates; this may be indicative of production of a hyperalgesic state caused by formalin injection. The results of this study support the concept that formalin injected into the lundpaw activates segmental antinociceptive systems which block SP release and hmlt nociceptive transmission. These results support evidence that SP may play a limited role in the response to formalin. INTRODUCTION The undecapeptide substance P (SP) is contained in the central terminals of small diameter primary afferent neurons which terminate in the superficial laminae of the dorsal horn of the spinal cord 19. Dorsal rhizotomy decreases dorsal horn SP content by 80%; the other 20% is contained in intrinsic or descending neurons 26. The A~ and C fibers containing SP can be activated by a number of noxious stimuli and respond by releasing SP into the dorsal horn 5'11'15'27-29'35'36'42. The actions of SP in the spinal cord are widely accepted as being involved in the transmission of nociceptive information 17'3s. Iontophoretic application of SP onto dorsal horn neurons has been shown to excite the same neurons as noxious peripheral stimulation 16. Furthermore, there is a large body of evidence associating intrathecal injections of SP with nociceptive behavior 24'39 and decreased nociceptive threshold 25, although there is some evidence to the contrary2,3,13,14. The formalin test has been widely accepted as a model of chemogenic pain 1°'22'41. The injection of dilute formalin into the paw of animals has been shown to produce a
biphasic response with both early and late phases of pain-related behavior 1°'22'41. Electrophysiological studies have demonstrated similar biphasic increases in the excitability of dorsal horn cells following formalin injection into their receptive fields 8'9. It has also been shown that the injection of formalin into the hindpaw as a nociceptive stimulus produces a biphasic increase in dorsal horn SP levels 32. These increases in dorsal horn SP-like immunoreactivity (SPLI) may be due to decreased SP release from primary afferent neurons 32. There is evidence that endogenous opiate systems are activated by the injection of formalin into the hindpaw 4'31'32. Both SP levels and release may be regulated by the primary afferent neuron during nociception. Much is known concerning the release of SP from tissue slices 3°' 36,37 or from the entire spinal cord in vivo 15'42. However, less is known about the release patterns of SP at specific sites in the terminal fields of small diameter primary afferent neurons when their specific receptive fields are activated. Satoh and his coworkers have performed several studies demonstrating the release of SP into the dorsal horn in both decerebrate/anesthetized rabbits and rats 27-29'35. This study was undertaken to determine the
Correspondence. B.D. Goldstein, Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300, U.S.A.
110
tern of S P L I in the superficial dorsal h o r n o f the l u m b a r
mg outflow was 2 8 - 0.26 pg SPLU100/zl. This corresponded to a recovery of 56% of the SP concentration in the standard solution.
spinal c o r d during the application o f a c u t e and s u b a c u t e
Radtotmmunoassay
n o c i c e p t i v e stimuli to the h i n d p a w s of rats.
The primary antisera was procured from Peninsula laboratories and supplied as lyophilized powder. A dilution was chosen that yielded approximately 45% specific binding of the 15,000 cpm of tracer added to each assay tube. The tracer used was 123I-[Tyr8] substance P as supphed by New England Nuclear. The assay buffer was a phosphate-buffered saline (pH 7.4) having the following composition: Na2HPO 4 9 raM, NaH2PO 4 41 raM, NaC1 0.14 M, bovine serum albumin 0.75%, and NaN 3 0.02%. Standards, blanks, and total binding tubes each received 200 #l of CSF m order to match unknown samples. Each tube was vortexed and allowed to incubate for at least 24 h at 4°C. An activated charcoai/dextran suspension was used to separate the free tracer. The resulting standard curve typically had an ICso of about 15 pg SP/assay tube and minimum detectable limit of about 1 pg SP/assay tube The primary antlsera cross-reacts with other SP related peptldes as follows: SP(5-11), 30%; SP-(3-11), 19%; SP-(7-11), 14%; SP-(1-4), less than 2.5%; and SP-(1-6), SP-(1-7), SP-(1-9), eledoisin, neurokmm A, neurokmin B, SP-GIy (fl-preprotachylonin)-(58-69)), and SP-GlyLys (fl-preprotachykinin-(58-70)) all less than 1% (all values calculated on a percentage molar basis).
effects o f n o c i c e p t o r activation o n the local release pat-
MATERIALS AND METHODS
Dorsal horn perfusion SPLI release was studied using a mochfication of a method described by Oku et al. as. Briefly, rats were anesthetized with halothane, immohilized with succinylcholine, and artificially respired. A dorsal laminectomy was performed to expose the lumbar enlargement The lammectomy was extended rostrally about 1 cm to provide a space where an acute spinal transection was performed. A carotid arterial cannula was inserted through which blood pressure was momtored throughout the experiment. The rat was then decerebrated at the level of the collicull and the anesthetic removed Following surgery, the rat was placed in a spinal unit and body temperature maintained with a thermistor-regulated heating pad placed under the animal. The pia mater was opened and a push-pull cannula devised from two 27-gauge needles was stereotaxacally introduced into the dorsal horn at the entry zone of the fifth lumbar dorsal root, 1 mm from the midline and approximately 700 #m deep. In each animal only one perfusion site was established. A peristaltic pump was used to introduce and remove artlficaal cerebrospinal fluid (CSF) through the cannula at a rate of 30 #l/min. The artificial CSF contained (in raM): NaC1 128.5, KCI 3.0, NaHCO a 21.0, NaHzPO 4 0.25, glucose 3.4, CaCI2 1.15, MgC12 0.8 and (in #M): bestatin 10, captopril 5, leupeptin 1.0, and chymostatin 1.0. The CSF was bubbled with 95% 02/5% CO2 to maintmn a pH of 7.4 and warmed to 37°(2 immediately before entering the cannula. Perfusion continued for at least 1 h before collection of samples for radiolmmunoassay (RIA) for SP. Perfusates were collected d~rectly into ~ce-cold assay tubes which contmned SP ant~sera. Samples (200/d) were collected into each assay tube every 6.66 rain. Following termination of the experiment, the location of the cannula tip in the dorsal horn was verified histologically. Animals m which the perfuston area was outside of laminae I and II of the dorsal horn or with cardiovascular events which altered SPLI release (such as blood pressure spikes or drops to less than 50 mm Hg) were excluded from the study.
Experimental protocol For each experiment, SPLI release was momtored for 60 mm prior to hindpaw treatment to establish a baseline control release rate for each ammal. The rats then received a 100 #1 injection of either 5% formalin or sterile isotonic saline into the plantar aspect of the hindpaw, or one of 3 intensities of mechanical pinch. Low and medium intensRy pinches were applied using small bulldog clamps with jaw opening weights of 50 and 250 g, respectively. Each of these pinches was delivered for 30 s each minute for 20 min to various loeatmns on the hindpaw and lower limb. A high intensity pinch was applied using a hemostatlc forceps w~th a jaw opening weight of approximately 1000 g applied once to the hindpaw for the entire 20-rain stimulus period. Jaw opening weights were measured by immoblhzmg one jaw of the forceps and hangmg weights from the lower jaw until the weight necessary to just open the forceps was reached. All stimuli were applied to the hindpaw lpsilateral to the site of dorsal horn perfuslon. Each expenmental subject received only one stimulus, with the exception of one group of animals in which the medmm intensity pinch was applied 90 min after formalin injeetaon. An experiment to determine the recovery of SP by the push-pull cannula was performed m which the cannula was immersed in a CSF solution containing SP and CSF moved through the cannula at the same rate as in animal experiments. The resulting cannula effluent was then assayed for SP content. When the push-pull cannula was inserted m a CSF containing 5 0 pg SP/100/zl, the result-
Data analysts SPLI release data were collected as pg SPLI/sample; m the figures, release data are shown as percent of basehne release and were compared to the sample period immediately preceding stimulus application. Thus, in the inset data are normalized to the 6.66-min period preceding hindpaw treatment; m the larger graph, data are normalized to the 20-min period preceding hmdpaw treatment. This longer baseline penod includes additional data not used in the reset Although this results in graphs which appear different, the raw data are identical for the treatment periods of each figure. Both graphs are included in order to show the nature of the raw data as collected (6.66-rmn intervals) and to facilitate the analysis of general trends in the data (20-min intervals). Analysis of covanance (ANCOVA) was used to test the raw data for statistical significance. Tukey's HSD test was used to perform post-hoc comparisons. Significance was set at P < 0.05, and all values are reported as the mean - S E.M
RESULTS Table I shows the n u m b e r of subjects u s e d for e a c h t r e a t m e n t and w h e t h e r t h e i r g e n e r a l t r e n d was t o w a r d i n c r e a s e d o r d e c r e a s e d S P L I r e l e a s e rates. T h e baseline S P L I release rates v a r i e d widely b e t w e e n e x p e r i m e n t a l subjects. T h e m e a n b a s e l i n e r e l e a s e r a t e was 6.63 --- 0.96 pg S P L I / s a m p l e (0.99 pg S P L I p e r m i n u t e ) . T h e variation of the 60-min b a s e l i n e p e r i o d f r o m s a m p l e to sample (6.66 m i n ) a v e r a g e d 12% of the b a s e l i n e that was used for the c o m p a r i s o n o f t h e n o c i c e p t i v e stimulus. Fig. 1 shows the r e l e a s e p a t t e r n of S P L I f o l l o w i n g application o f t h e low intensity pinch to the h i n d p a w . This pinch was mildly n o x i o u s w h e n a p p l i e d to t h e w e b b i n g b e t w e e n the i n v e s t i g a t o r ' s fingers. It can be s e e n in Table I that this pinch p r o d u c e d i n c r e a s e d S P L I release in half of t h e recipients. In the rat it p r o d u c e d a 17% increase in the a m o u n t of S P L I r e l e a s e d into the ipsilateral L 5 dorsal h o r n during the 20 m i n of stimulus application (Fig. 1). W h e n v i e w e d in 6.66-min samples (see
111 TABLE I Changes in SPLI release in individual subjects Treatment
n
Low intensity pinch 6 Medium intensity pinch 13 High intensity pinch 3 Formahn 6 Saline 7 Medium mtenslty pinch after formahn 4
Number (%) of ammals showing:* Increase
Decrease No change
3 (50%) 12 (92%) 3 (100%) 0 0
0 1 (8%) 0 6 (100%) 7 (100%)
4 (100%) 0
3 (50%) 0 0 0 0 0
* Mean of SPLI release values during treatment (20 min for pinches, 80 min for formalin and saline) compared to mean of 20 min baseline for same experimental subject. inset), it can be seen that SPLI release was increased by as much as 110%. This increase was significantly different from baseline release only when the d a t a were l u m p e d into 20-min periods. T h e differences in a p p e a r ance of the d a t a shown in each figure and its inset are due to the calculation of percent of control values with respect to different periods of baseline release values (see Materials and Methods). Fig. 2 shows the release p a t t e r n of SPLI into the dorsal horn following application of the m e d i u m intensity pinch to the hindpaw. This pinch was frankly noxious
A lOO
B 2O0
when applied to the investigator, and Table I shows that it p r o d u c e d increased SPLI release in 92% of recipients. It p r o d u c e d an increase in SPLI release which was greater in both intensity and duration than the low intensity pinch (Fig. 2). W h e n viewed in 20-min periods, SPLI release increased to 33% of baseline rates during the application of the stimulus, r e m a i n e d elevated (26%) during the 20 min following termination of the stimulus, and r e t u r n e d to baseline 20 min after termination of the stimulus. The inset shows that when viewed in 6.66-rain periods, similar increases were seen (25-30%) during the application of the stimulus, but SPLI release was not significantly different from baseline release rates as soon as the stimulus was terminated. The effects of the high intensity pinch are shown in Fig. 3. This pinch p r o d u c e d inflammation and tissue injury in the hindpaw of the rat and was not tested by application to the investigator. Table I shows that all animals receiving the high intensity pinch r e s p o n d e d with increased SPLI release. It p r o d u c e d a 146% increase in SPLI release during the 20-min stimulus period; when viewed in 6.66-rain periods the increase was as great as 587% (Fig. 3). This increase was significant c o m p a r e d to both 6.66- and 20-min baseline periods; however, the increase was short-lived c o m p a r e d to the m e d i u m intensity pinch. L o o k i n g at the inset, it can b e seen that release r e t u r n e d to slightly below baseline immediately following termination of the pinch. The time course of SPLI release following formalin injection into the hindpaw is shown in Fig. 4. F o r m a l i n p r o d u c e d a significant decrease in the release of SPLI.
I
L
0
~-g
i
h
20
1 Low
Intensity
40 Pinch
50
A
B
100
..=
g
, 60
~ 50 E O
o -20 Low IntensntyPinch
i i i 0 20 40 I Medium Intenstty Pinch
-50
0 T
1
20 40 Time (minutes)
Fig. 1. Effects of low intensity pmch on SPLI release. Graphs show the time course of SPLI release in the dorsal horn of animals receiving a low intensity noxious pinch applied to the hindpaw (n = 6). The pinch was applied during the time period denoted by the cross-hatched area of the graph. B shows data resulting from 6.6re.in sample periods and percentages of control are compared to the one 6.6-rain sample period immediately preceding stimulus application. In A, each bar represents the same data lumped into 20rain sample periods for clarity; percents of control are then based on the mean of the three 6.6-rain sample periods immediately preceding application of the stimulus. *P < 0.05 as compared to baseline release (analysis performed on raw data).
I g
o
-20 Medium Intensity Pinch
I
0 T
20
40 60 Time (minutes)
Fig. 2. Effects of medium intensity pinch on SPLI release. Graphs show the time course of SPLI release in the dorsal horn of animals receiving a medium intensity noxious pinch applied to the hindpaw (n = 13). The pinch was applied during the time period denoted by the cross-hatched area of the graph. Data are reported as in Fig. 1.
112
B
A,oo
B 1000
0 ~
I
5
-50 l
_= 50 •" 400
0 20 l H i g h Intensity P m c h
-100 [
40
0
20 rSahne
40
60
80
©
gE
o
0
e1
~ -50
o
-100
-20 H~gh Intensity Pinch
0 T
.... -20
20 40 Ttme (minutes)
20
40
Sahne
F~g. 3. Effects of high-intensity pinch on SPLI release. Graphs show the time course of SPLI release in the dorsal horn of animals receiving a high intenmy noxious pinch applied to the hindpaw (n = 3). The pinch was apphed during the time period denoted by the cross-hatched area of the graph. Data are reported as m Fig. 1.
It can be seen in the inset that formalin produced a biphasic effect, with SPLI release being inhibited by up to 60% for the first 40 min after injection into the hindpaw and again after 60 rain following formalin injection. After 80 min SPLI release remained suppressed in experiments which were not terminated by that time (data not shown for clarity). The effects of the control injection of saline are shown in Fig. 5. Although there was a trend toward saline decreasing SPLI release, none of the effects were significantly different from baseline release rates when analyzed in 6.66- or 20-rain intervals. Table I
60 80 Time (minutes)
Fig. 5. Effects of saline on SPLI release. Graphs show the ume course of SPLI release in the dorsal horn of ammals recelvang an rejection of 100/~! of sahne into the hindpaw (n = 7). Data are reported as in Fig. 1.
shows that both saline and formalin produced decreased SPLI release in all subjects. Fig. 6 shows the effect of the medium intensity pinch when applied approximately 90 min following formalin injection into the hindpaw. It can be seen that during application of the pinch SPLI release was increased to a degree similar to that in normal animals in both 20- and 6.66-min periods (see Fig. 2). However, in the formalin injected animals SPLI release did not return to baseline rates; instead, SPLI release progressively increased to as
B
A I000
AI®
1ooo ;
-
I
-50
~" e~
0
50
100
I) r
- -
0 20 40 60 T M e d i u m lntenstty Pinch After F o r m a h n
500 t 0 20 l Formahn
40
60
80
O
E
~
-50
T -20 0 Medtum IntensRy, Pinch t Atter rormaun /
-100
-20
0 20 T Fonnahn
40
60 8(1 Time (minutes)
Fig. 4 Effects of formahn on SPLI release. Graphs show the time course of SPLI release in the dorsal horn of ammals recewmg an rejection of 100/~1 of formahn into the hindpaw (n = 6). Data are reported as m Ftg 1
20
40 60 Time (minutes)
Fig. 6. Effects of medium intensity pinch after formahn on SPLI release. Graphs show the time course of SPLI release in the dorsal horn of animals recewmg a medium intensity noxious pinch applied to the hmdpaw 90 mm after injection of 100/A of formalin into the same lundpaw (n = 4). The pinch was applied during the time pen o d denoted by the cross-hatched area of the graph. Data are reported as in Fig. 1
113 much as 389% (20-min period) or 1400% (6.66-min period) following termination of the stimulus. These increases were only significant when analyzed in 20-min periods. Table I shows that application of the medium intensity pinch after formalin uniformly produced increases in SPLI release. DISCUSSION Much information has been gained concerning the release of SP in the spinal cord using techniques such as perfusion of tissue slices3°'36'37 or the entire spinal cord 15' 42. Such methods are limited in their ability to localize the specific site of SP release. Antibody-coated microprobes 11 and microdialysis probes 5 have been successfully used to localize SP release but may be limited in their ability to quantify the amount of peptide released 7. The stereotaxic push-pull cannula used in this study and studies by other investigators 27-29'35 can localize not only the specific site but also the quantity of released neurotransmitters. There is evidence that push-pull perfusion techniques may yield much higher recovery of peptides than microdialysis techniques 7. The recovery of 56% of a perfused solution in this study demonstrates the ability of the push-pull cannula to assess peptide content with a degree of accuracy which may not be possible with techniques which rely on diffusion of peptides through a dialysis membrane. Here we report the release patterns of SPLI into the L5 superficial dorsal horn following the application of various nociceptive stimuli to the hindpaw of unanesthetized decerebrate/spinal rats. It is likely that the majority of the SPLI released into the dorsal horn is actual substance P. N-terminal SP fragments are found in rat CNS 4°, and may have biological activity6, but show very little crossreactivity toward the SP antisera used in this study. The primary antisera only cross reacts, to any appreciable degree, with C-terminal SP fragments, primarily SP-(5-11) (30%). The vast majority of SP C-terminal fragments found in the rat CNS coelute with SP-(511) in gel electrophoresis4°, and a metabolically protected SP-(5-11) analogue 12 has been shown to have biological activity in rat CNS. However, it has been shown that pyroglutamicS-SP-(5-11) (the naturally occurring cyclized metabolite of SP-(5-11) is not present in the potassium stimulated release products of isolated rat spinal cord 1. Thus, SP-(5-11) probably does not contribute to the SPLI measured in these studies. Primary afferent neurons which contain SP are probably the major source of the SPLI release measured in this study. Most of the SP content of the dorsal horn is contained in primary afferent terminals 26. The superficial dorsal horn areas perfused in this study correspond
with the terminal fields of small diameter primary afferent neurons which contain SP 19. The contribution of evoked SPLI release from descending neurons is unlikely in the spinal transected animals used in this study. Furthermore, other investigators have shown that the SP content of dorsal horn tissues remains unchanged when intrinsic neurons are destroyed by the occlussion of the spinal cord blood supply21. The increased SPLI release seen during noxious mechanical stimulation is consistent with results of other studies 15'29. This study also demonstrates a graded response of increasing SPLI release with increased intensity of the pinch applied to the hindpaw. There is a distinct increase in the magnitude and duration of the evoked SPLI release between the low and medium intensity noxious pinches; this is consistent with the activation of nociceptive afferent neurons. The high intensity pinch showed a further increase in the amount of SPLI released, but the duration of the response was attenuated. This intense stimulus produced inflammation and tissue damage which may have activated systems which respond to limit SPLI release, an effect similar to that produced by formalin. Alternatively, the high intensity pinch may have simply depleted the terminal content of SP available for release into the dorsal horn. In this study, formalin clearly produced a decrease in the amount of SPLI released into the dorsal horn. The physiology of the changes induced by formalin is poorly understood, but there is evidence that an endogenous opiate system plays a role in the control of the transmission of nociceptive information. The intense nociceptive activation produced by formalin activates opiate antinociceptive systems which act to produce a buildup of SPLI content in the dorsal horn 31'32. Although the effects of descending inhibitory systems have been removed in these experiments by acute spinal transection, the existence of enkephalinergic segmental control systems activated by noxious stimuli has been demonstrated 43. The segmental release of Met-enkephalin has been shown to increase following formalin injection into the hindpaw 4. Additionally, the opiate antagonist naloxone has been shown to increase SP release 15 and potentiate the pain-related behavior evoked by noxious stimuli41. Thus, there is evidence that the dynamics of the responses of SP to nociceptive stimuli are in part under the control of a system of endogenous opioid peptides that limit the transmission of nociceptive information. This study provides evidence that these systems may act by blocking the release of SP from primary afferent neurons after an intense stimulus such as formalin injection. In this study, no increases in SPLI release were seen following formalin injection such as those described by Kuraishi et al. 29. It is, of course, possible that there is
114 an initial increase in SPLI release within the first sample collected which is of short enough duration to be masked by decreased release for the remainder of the sample period. However, we feel this is unlikely given the magnitude of the decrement in SPLI release evident in the samples immediately following formalin injection. Interestingly, saline injection into the hindpaw also tended to decrease SPLI release, but not to a significant degree. It is possible that the needle prick itself is sufficient to midly activate the antinociceptive systems which decrease SPLI release in response to formalin. Alternatively, the volume of the injection may activate lowthreshold primary afferent systems which act to limit the release of SP and the transmission of nociceptive information. Gate control systems activated by non-noxious stimuli have been proposed 33, but there is evidence that A fiber electrical stimulation does not decrease SPLI release evoked by noxious stimuli 23. It is also shown in this study that the application of the same intensity of noxious pinch to normal and formalin injected animals produces markedly different patterns of SPLI release. The progressive increases in SPLI release following termination of the pinch may reflect a hyperalgesic state of nociceptors in the hindpaw produced by the injection of formalin. Clearly, whereas SPLI release returns to baseline rates soon after the termination of noxious stimulation of normal hindpaws, SPLI release remains elevated and even increases further when the same stimulus is applied to formalin-injected hindpaws. A previous report by Kuraishi, et al. 29 has shown increases in SPLI release lasting exactly 20 rain in response to all the types of nociceptive stimuli used, including pinch, intense heat and formalin. In this study we demonstrate similar increased SPLI release due to noxious mechanical stimulation, but marked long-lasting de-
creases in the SPLI release following injection of formalin into the hindpaw. The studies conducted by Kuraishi et al. used similar methods, but were conducted using decerebrate/anesthetized rabbits with intact descending control systems and the possibility of anesthetic effects 2729. Either of these differences from the preparation used in this study could account for the differences seen in the response to formalin. Both pentobarbital and urethane anesthesia masked evoked SPLI release in our preparation (unpublished observations). We feel that the effects of formalin on release in this study may be more consistent with its long-term effects on nociceptive behavior 1°'22'41, dorsal horn neuron excitability s'9, and dorsal horn SPLI content 31'32. Finally, it must be considered that formalin may not be a purely nociceptive agent when used to activate SP containing neuronal systems. Increases in dorsal horn SPLI content stimulated by formalin can be attenuated by administration of naloxone 31'32, yet naloxone has little, if any, effect on formalin-induced nociceptive responses 18'24. Similarly, blockade of formalin-induced nociceptive behaviors by pretreatment with lidocaine does not reduce the concomitant increases in dorsal horn SPLI content 2°. Also, intrathecal injections of an SP antagonist or SP antiserum inhibit nociceptive behavior only in the early phase of the formalin response 34. Taken together with the present demonstration that SPLI release is suppressed at the same time that nociceptive behavior would be elevated, the evidence seems to suggest that SP may not be directly involved in the actual transmission of nociceptive information at least during the late phase of the formalin test.
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