Brain Research. 542 (1991) 212-218
212
Elsevicr BRES 16354
Neonatal capsaicin treatment abolishes the nociceptive responses to intravenous 5-HT in the rat S.T. Meller 1, S.J. Lewis ~, T.J. Ness 1'2, Michael J. Brody* and G.F. Gebhart 1 Departments of tPharmacology and 2Anesthesia, College of Medicine, University of Iowa, Iowa City, IA 52242 (U.S.A.) (Accepted 11 September 1990)
Key words: Serotonin; Intravenous; Nociception; Neonatal capsaicin; Rat
The intravenous (i.v.) administration of serotonin (5-HT) to lightly pentobarbital-anesthetized rats is known to produce a triad of reflex cardiovascular responses, distinct afferent-mediated pseudaffective reactions, and a vagally mediated inhibition of the nociceptive tail-flick (TF) reflex consistent with 5-HT acting as a noxious stimulus. In the present experiments we examined the involvement of capsaicin-sensitive afferents in mediating these responses. Lightly pentobarbital-anesthetized 16-week-old male Sprague-Dawley rats which had been treated as neonates (in the first 48 h of life) with capsaicin (50 #g/kg, s.c.) were compared to age-matched neonatal vehicle-treated controls. Whereas the i.v. administration of 5-HT produced a dose-dependent (6-96 btg/kg, i.v.) inhibition of the nociceptive TF reflex (EDs0 = 48.1 + 11.3/ag/kg; n = 7) and distinct pseudaffective responses (usually by 24-48 btg/kg) in vehicle-treated rats, 5-HT (6-192/,g/kg, i.v.) failed to significantly alter TF latency or produce pseudaffective behaviors in the capsaicin-treated rats (n = t0). There was no difference in baseline TF latencies between the two groups. There were essentially no differences between vehicle- and capsaicin-treated rats with respect to the initial cardiopulmonary vagal afferent-mediated (Bezold-Jarisch reflex) decreases in heart rate and arterial blood pressure or the subsequent pressor phase. However, the magnitude of the late hypotensive phase was significantly greater in capsaicin-treated rats. Capsaicin-treated rats were significantly heavier than vehicle-treated rats (482.0 + 12.4 g vs 439.3 + 12.1 g) and also exhibited small cutaneous lesions around the nostrils, on the skin in and around the vibrissae, behind and on the back of the ears, on the throat, and on the underside of the belly. Radioimmunoassay showed that there was a significant (P < 0.05) depletion of substance P (58.3%) and calcitonin gene-related peptide (50.7%) in the lumbar spinal cord of neonatal capsaicin-treated rats (n = 8) compared to vehicle-treated rats (n = 5). These results demonstrate that the 5-HT-induced, vagally-mediated inhibition of the TF reflex and pseudaffective responses are mediated by capsaicin-sensitive afferents. This strongly suggests that (1) afferent fibers subserving the nociceptive response are predominantly unmyelinated C-fiber vagal afferents and (2) these afferents are different from those vagal afferents that mediate the cardiovascular responses to i.v. 5-HT.
INTRODUCTION
tent. Either increases 11'12'14'17'19'25'36'37'42 decreases 7'19, or no change 4'5j1'14'18'2°'21 in thermal, mechanical or chemical
The administration of capsaicin (8-methyl-N-vanillyl6 - n o n e n a m i d e ) to neonates is r e p o r t e d to destroy up to 35827 95% of u n m y e l i n a t e d C-fiber sensory afferents . . . . 36,38,41 C o n c o m i t a n t with these morphological changes are m a r k e d reductions in the contents of substance P (sP), calcitonin gene-related peptide ( C G R P ) , vasoactive intestinal polypeptide, somatostatin, cholecystokinin (CCK) and o t h e r peptides in the spinal cord, nucleus of the solitary tract (nTS), dorsal root ganglia and nodose ganglia (NG) (for reviews, see refs. 3, 44). In addition, neonatal administration of capsaicin has been r e p o r t e d to result in alterations in cardiovascular, p u l m o n a r y , t h e r m o r e g u l a t o r y , gastrointestinal, bladder, ocular, inflammatory and nociceptive sensory functions (for reviews, see refs. 3, 32, 44). However, the effects of this treatment on cutaneous nociceptive functions are inconsis-
nociceptive thresholds have been reported. In contrast, previous reports have consistently demonstrated a marked elevation of visceral nociceptive thresholds 1'6 and an almost complete abolition of stimulus-induced behavioral reactions 1 following neonatal capsaicin-treatment. The intravenous (i.v.) administration of serotonin (5-HT) is r e p o r t e d to be painful in h u m a n s 23 and produces pseudaffective responses in rats 33"34 and m o n k e y s 2. The responses in rats are m e d i a t e d largely through activation of c a r d i o p u l m o n a r y vagal afferents 3°' 33,34. H o w e v e r , as i.v. 5-HT also produces m a r k e d , vagally m e d i a t e d , cardiovascular changes, it is not clear w h e t h e r the vagal afferents that m e d i a t e the nociceptive responses to 5 - H T are different from those that m e d i a t e the acute changes in cardiovascular function. In the present study, we r e p o r t that n e o n a t a l capsaicin
* Deceased 3 December 1990. Correspondence: S.T. Meller, Department of Pharmacology, Bowen Science Building, College of Medicine, University of Iowa, Iowa City, IA 52242-1194, U.S.A. 0006 8993/91/$03.50© 1991 Elsevier Science Publishers B.V. (Biomedical Division)
213 treatment
abolishes
the
5-HT-induced
pseudaffective
b e h a v i o r s a n d t h e 5 - H T - i n d u c e d i n h i b i t i o n o f t h e nocic e p t i v e tail-flick ( T F ) r e f l e x w i t h o u t a l t e r i n g t h e v a g a l l y mediated
5-HT-induced
Bezold-Jarisch
results suggest that small diameter, vagal
afferents
convey
the
reflex.
These
capsaicin-sensitive
nociceptive,
but
not
the
c a r d i o v a s c u l a r , r e s p o n s e t o i.v. 5-HT. MATERIALS AND METHODS
Animals A total of 17 male Sprague-Dawley rats (Biolabs, St. Paul, MN) were used in this study. Ten rats were treated in the first 48 h of life by a subcutaneous (s.c.) injection of 50 mg/kg eapsaicin (98%, lot no. 58F-7125; Sigma Chemical Co., St. Louis, MO) dissolved in saline vehicle containing 10% Tween 80 and 10% ethyl alcohol. Seven rats were also injected s.c. in the first 48 h of life with vehicle only. All animals were housed in a room maintained at a constant temperature of 22 *C on a 12/12 h light/dark cycle with food and water provided ad libitum. The groups are summarized in Table I.
Surgery All adult (16 weeks of age) rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (Nembutal, 45 mg/kg; Abbott Labs., IL) and the left femoral artery and vein were cannulated for the subsequent recording of arterial blood pressure and administration of drugs, respectively. The lines were externalized between the shoulder blades and all wounds were sutured and the margins were liberally coated with a local anesthetic ointment (Nupercainal, Ciba, Edison, NJ). The animals were allowed to recover from surgery for 3-7 days before experimental use (see below).
Nociceptive testing Nociceptive responses were determined using a TF apparatus as described previously33. Briefly, the apparatus consisted of a beam of focused radiant heat provided by a 50 W projector lamp. Radiant heat was focused systematically on the underside of the tail at one of 5 sites 8-10 mm apart with the most distal site approx. 4 cm from the tip. The TF latency was measured to the nearest 0.1 s as the time from onset of heating of the tall to withdrawal of the tail from the beam. A cut-off latency of 7 s was used to avoid tissue damage to the tail 39.
Experimental design Rats in the capsaiein-treated (n = 10) or vehicle-treated (n = 7) groups were given a single bolus dose of sodium pentobarbital (4.5-9.0 mg, i.v.) and allowed to recover to a light level of anesthesia (corneal and flexion withdrawal reflexes intact). In this comparatively light level of anesthesia, nociceptive withdrawal reflexes can be evoked without producing complex coordinated
escape behaviors39. The rheostat on the TF apparatus was adjusted such that at 7 s, the temperature as measured by a thermometer bulb was 65 *C. Baseline "IT lateneies were randomly recorded 3 times at each of 5 different sites approx. 8-10 mm apart for a total of 15 trials. The time between successive trials was 30 s. The rheostat was then adjusted such that at 7 s the temperature as measured by a thermometer bulb was 45 °C or 55 °C. The 15 trials were then repeated at each temperature as outlined above. The heat was then readjusted to 65 °C such that the baseline TF latency was usually between 1.8 and 2.4 s and 5-HT creatinine sulphate (Sigma Chemical Co., St. Louis, MO) was administered in an ascending dose-dependent manner (6, 12, 24, 48, 72, 96, 144 or 192/zg/kg, i.v.) until inhibition of the TF reflex to cutoff (7 s). All subsequent measurements of TF latency were made at the fastest rate of heating. Animals were tested 3, 33, 63 and 123 s following the i.v. administration of 5-HT. Latency measurements were continued at two-min intervals thereafter until the animal returned to the baseline latency established pre-drug. Arterial blood pressure and heart rate (HR) were monitored continuously throughout the experiments. The rat's behavioral reactions to each dose of i.v. 5-HT were assessed qualitatively as either present or absent 33. Two to three days following determination of the dose-response curve to i.v. 5-HT, the animals from each group were briefly anesthetized with ether, decapitated, the sacral spinal cord at the level of the iliac crest was severed with sharp scissors, and the spinal cords were extracted by hydraulic extrusion using a 16 gauge needle inserted into the spinal cord and flushed with 10 ml saline. The spinal cord was removed, wrapped in foil, and immediately frozen in liquid nitrogen. These samples were stored at -70 °C and subsequently a 2-cm piece of lumbar spinal cord, including the lumbar enlargement, was processed for sP and CGRP radioimmunoassay (RIA).
sP and CGRP RIA The content of sP and CGRP in each spinal cord was quantified by RIA. The lumbar enlargements of the frozen spinal cords were minced, homogenized and placed in boiling 0.1 N hydrochloric acid for 15 min and subsequently centrifuged at 12,000 g for 10 rain. Aliquots of the supernatant were. frozen for subsequent RIA. For sP, the samples were thawed, lyophilized and preincubated with antibody for 16 h at 4 *C. IlZLlabelled TyrS-sp containing 4% normal rabbit serum was added and incubated for an additional 24 h at 4 °C. The bound sP was separated from the free sP by the addition of a secondary goat anti-rabbit antibody. The final antibody concentration was 1:120,000 and the assay buffer consisted of 0.04 M KH2PO4, 1% BSA, 0.25% trasylol and 0.02% thimerosal, pH 6.5 with 4% PEG-8000 and 0.01 M EDTA. The absolute sensitivity of the assay was 10 pg/ml. For CGRP, the samples were preincubated for 16 h at 4 °C. I125-1abelled tyr°-CGRP (rat) was then added and incubated for an additional 16 h at 4 °C. Bound CGRP was separated from the free CGRP by the addition of a secondary donkey anti-goat antibody. The final antibody concentration was 1:84,000 and the assay buffer consisted of 0.06 M Na2HPO4, 0.013 M EDTA, 0.02% sodium azide, 0.1% triton-X, pH 7.4. The absolute sensitivity of the assay was 150 pg/ml. In either assay the inter- and intra-assay variation was less than 10%.
TABLE I
Data acquisition and statistical analyses
Summary of experimental groups Neonatal treatment
Number (n) Weight (g) Resting MAP (mmHg) Resting HR (b.p.m.) Baseline TF (s)
Capsaicin
Vehicle
10 482 + 12" 111 + 4 349 + 10 2.4 + 0.1
7 439 + 12 112 + 5 339 + 17 2.1 + 0.1
* Significantly different from vehicle-treated rats, P < 0.05.
All data are presented as means + S.E.M. The mean TF latencies are presented as raw data in seconds. Raw TF data were analyzed by sigmoidal curve fitting analysis which generated the ED5o and the gain of the 5-HT-mediated inhibition of the TF reflex. All other data were analyzed by analysis of variance with covariance (ANCOVA) 5°. Differences between individual means were determined by Student's modified t-test with a Bonferroni correction for multiple comparisons utilizing the modified error mean squared terms from the covariance analysis49. Correlations between inhibition of the TF reflex at the greatest doses of 5-HT used, and spinal contents of sP or CGRP were made with Spearman's rank correlation procedure for the capsaicin-treated rats. To standardize
214 comparisons, TF values were expressed as TF index (TFI) which is calculated as: (trial latency-baseline latency) divided by (cutoff latency-baseline latency) multiplied by 100 and expressed as a percentage depletion of p~ptide compared to the mean of vehicletreated rats.
7
0 neonatal vehicle • neonatal capsaicin
0
I
_/
I/
RESULTS
2 Comparison o f baseline T F latencies at different rates o f skin heating As the reported effects of neonatal capsaicin on heat-evoked nociceptive reflexes are inconsistent 4'7'n' 14,18-21,25,36,37, the rate of heating of the tail in this study was adjusted such that at cutoff latency (7 s) the temperature of the beam reached either 45, 55 or 65 °C. Therefore, any differences between the capsaicin-treated and vehicle-treated groups might readily become apparent. However, there was no difference in baseline TF latencies between capsaicin-treated rats and vehicletreated rats at any of the 3 rates of heating (Table II). Although, in both the capsaicin-treated and vehicletreated rats, there was a positive correlation between the rate of tail heating and TF latency; that is, TF latencies were significantly faster at the higher rate of heating. Effect o f i.v. 5 - H T TE 5-HT (6-96/~g/kg, i.v.) produced a dose-dependent inhibition of the TF reflex (n = 7) in vehicle-treated rats. The EDs0 (50% of maximal effect) for inhibition of the TF reflex was 48.1 + 11.3 /~g/kg. In contrast to vehicle-treated rats, 5-HT (6-192/~g/kg, i.v.) did not, as a group, significantly alter TF latency from baseline in capsaicin-treated rats (n = 10) (Fig. 1). In 6/10 capsaicintreated rats, 5-HT did not alter T F latency even at the greatest dose administered (192/~g/kg, i.v.); however, 3/10 of the capsaicin-treated rats exhibited slightly increased TF latencies at the greatest doses tested and 1/10 of the capsaicin-treated rats was completely inhibited to cut-off by 144 /,g/kg 5-HT. Baseline TF latency in capsaicin-treated rats was not significantly different from vehicle-treated rats (Table II). Cardiovascular. In both vehicle- (6-96 ~ug/kg, i.v.) and
TABLE II
~
~o
•-*-I ~
I
0
I B
, 3
10
, 30
100
300
dose 5-HT ( ~ g / k g )
Fig. 1. Alteration in the latency of the nociceptive tail-flick (TF) reflex produced by graded doses of intravenous (i.v.) serotonin (5-HT) in neonatal capsaicin-treated (0) (n = 10) or vehicle-treated rats (O) (n = 7). The dose administered is represented on the x-axis in pg/kg and changes in TF latency are represented on the y-axis in seconds. All data points are expressed as means + S.E.M.I.v. 5-HT produced a significant (P < 0.05) dose-dependent inhibition of the TF reflex (EDs0 = 48.1 + 11.3/~g/kg, gain = 22.3 + 7.5 s/log10dose; P < 0.05), whereas 5-HT administered to capsaicin-treated rats did not produce any significant change in TF latency (P > 0.05 for all comparisons).
capsaicin- (6-192 #g/kg, i.v,) treated rats, 5-HT typically produced a dose-related triphasic effect on arterial blood pressure and a bradycardia. The cardiovascular responses consisted of a brief, Bezold-Jarisch reflexly mediated bradycardia and associated decrease in arterial blood pressure followed by an increase in arterial blood
A
Neonatalvehicle
"~ 200]
DJ
rff Oj
B
T
T
T
30 sec
Neonatalcapsaicin
200]
5001
Summary of baseline TF latency at different rates of heating Neonatal treatment Vehicle Capsaicin a
Temperature at 7 s
Ou
45 °C
55 °C
65 °C
3.5 + 0.1 3.9 + 0.2
2.6 +_.0.1" 2.8 + 0.1"
2.1 + 0.1" 2.4 + 0.1"
a No difference between capsaiein- and vehicle-treated rats at all temperatures, P > 0.05. * Significantly different from 45 °C, P < 0.05.
T
12 pg/kg
T
4-8
pg/kg
T
72
pg/kg
Fig. 2. Illustration of typical pulsatile arterial blood pressure (PP; mmHg) and heart rate (HR; b.p.m.) responses to 5-HT (12, 48 or 72/~g/kg, i.v.) in (A) neonatal vehicle-treated rats or (B) neonatal capsaicin-treated rats. 5-HT was administered in the femoral vein. The time of the injection is indicated by the large arrow. Note the similarity in blood pressure and heart rate responses to 5-HT in neonatal vehicle- and capsaicin-treated rats.
215
A
0
•
vehicle n
-50 E
C
50 il
~
40
-100
",--" - 1 5 0 tic -200
~ 20 ~ lO
-250 -500
i
0
i
B
0
i
D
0
-10
i
-10
-20 -20 E
-30
-30
< -40 ,
212
Elsevicr BRES 16354
Neonatal capsaicin treatment abolishes the nociceptive responses to intravenous 5-HT in the rat S.T. Meller 1, S.J. Lewis ~, T.J. Ness 1'2, Michael J. Brody* and G.F. Gebhart 1 Departments of tPharmacology and 2Anesthesia, College of Medicine, University of Iowa, Iowa City, IA 52242 (U.S.A.) (Accepted 11 September 1990)
Key words: Serotonin; Intravenous; Nociception; Neonatal capsaicin; Rat
The intravenous (i.v.) administration of serotonin (5-HT) to lightly pentobarbital-anesthetized rats is known to produce a triad of reflex cardiovascular responses, distinct afferent-mediated pseudaffective reactions, and a vagally mediated inhibition of the nociceptive tail-flick (TF) reflex consistent with 5-HT acting as a noxious stimulus. In the present experiments we examined the involvement of capsaicin-sensitive afferents in mediating these responses. Lightly pentobarbital-anesthetized 16-week-old male Sprague-Dawley rats which had been treated as neonates (in the first 48 h of life) with capsaicin (50 #g/kg, s.c.) were compared to age-matched neonatal vehicle-treated controls. Whereas the i.v. administration of 5-HT produced a dose-dependent (6-96 btg/kg, i.v.) inhibition of the nociceptive TF reflex (EDs0 = 48.1 + 11.3/ag/kg; n = 7) and distinct pseudaffective responses (usually by 24-48 btg/kg) in vehicle-treated rats, 5-HT (6-192/,g/kg, i.v.) failed to significantly alter TF latency or produce pseudaffective behaviors in the capsaicin-treated rats (n = t0). There was no difference in baseline TF latencies between the two groups. There were essentially no differences between vehicle- and capsaicin-treated rats with respect to the initial cardiopulmonary vagal afferent-mediated (Bezold-Jarisch reflex) decreases in heart rate and arterial blood pressure or the subsequent pressor phase. However, the magnitude of the late hypotensive phase was significantly greater in capsaicin-treated rats. Capsaicin-treated rats were significantly heavier than vehicle-treated rats (482.0 + 12.4 g vs 439.3 + 12.1 g) and also exhibited small cutaneous lesions around the nostrils, on the skin in and around the vibrissae, behind and on the back of the ears, on the throat, and on the underside of the belly. Radioimmunoassay showed that there was a significant (P < 0.05) depletion of substance P (58.3%) and calcitonin gene-related peptide (50.7%) in the lumbar spinal cord of neonatal capsaicin-treated rats (n = 8) compared to vehicle-treated rats (n = 5). These results demonstrate that the 5-HT-induced, vagally-mediated inhibition of the TF reflex and pseudaffective responses are mediated by capsaicin-sensitive afferents. This strongly suggests that (1) afferent fibers subserving the nociceptive response are predominantly unmyelinated C-fiber vagal afferents and (2) these afferents are different from those vagal afferents that mediate the cardiovascular responses to i.v. 5-HT.
INTRODUCTION
tent. Either increases 11'12'14'17'19'25'36'37'42 decreases 7'19, or no change 4'5j1'14'18'2°'21 in thermal, mechanical or chemical
The administration of capsaicin (8-methyl-N-vanillyl6 - n o n e n a m i d e ) to neonates is r e p o r t e d to destroy up to 35827 95% of u n m y e l i n a t e d C-fiber sensory afferents . . . . 36,38,41 C o n c o m i t a n t with these morphological changes are m a r k e d reductions in the contents of substance P (sP), calcitonin gene-related peptide ( C G R P ) , vasoactive intestinal polypeptide, somatostatin, cholecystokinin (CCK) and o t h e r peptides in the spinal cord, nucleus of the solitary tract (nTS), dorsal root ganglia and nodose ganglia (NG) (for reviews, see refs. 3, 44). In addition, neonatal administration of capsaicin has been r e p o r t e d to result in alterations in cardiovascular, p u l m o n a r y , t h e r m o r e g u l a t o r y , gastrointestinal, bladder, ocular, inflammatory and nociceptive sensory functions (for reviews, see refs. 3, 32, 44). However, the effects of this treatment on cutaneous nociceptive functions are inconsis-
nociceptive thresholds have been reported. In contrast, previous reports have consistently demonstrated a marked elevation of visceral nociceptive thresholds 1'6 and an almost complete abolition of stimulus-induced behavioral reactions 1 following neonatal capsaicin-treatment. The intravenous (i.v.) administration of serotonin (5-HT) is r e p o r t e d to be painful in h u m a n s 23 and produces pseudaffective responses in rats 33"34 and m o n k e y s 2. The responses in rats are m e d i a t e d largely through activation of c a r d i o p u l m o n a r y vagal afferents 3°' 33,34. H o w e v e r , as i.v. 5-HT also produces m a r k e d , vagally m e d i a t e d , cardiovascular changes, it is not clear w h e t h e r the vagal afferents that m e d i a t e the nociceptive responses to 5 - H T are different from those that m e d i a t e the acute changes in cardiovascular function. In the present study, we r e p o r t that n e o n a t a l capsaicin
* Deceased 3 December 1990. Correspondence: S.T. Meller, Department of Pharmacology, Bowen Science Building, College of Medicine, University of Iowa, Iowa City, IA 52242-1194, U.S.A. 0006 8993/91/$03.50© 1991 Elsevier Science Publishers B.V. (Biomedical Division)
213 treatment
abolishes
the
5-HT-induced
pseudaffective
b e h a v i o r s a n d t h e 5 - H T - i n d u c e d i n h i b i t i o n o f t h e nocic e p t i v e tail-flick ( T F ) r e f l e x w i t h o u t a l t e r i n g t h e v a g a l l y mediated
5-HT-induced
Bezold-Jarisch
results suggest that small diameter, vagal
afferents
convey
the
reflex.
These
capsaicin-sensitive
nociceptive,
but
not
the
c a r d i o v a s c u l a r , r e s p o n s e t o i.v. 5-HT. MATERIALS AND METHODS
Animals A total of 17 male Sprague-Dawley rats (Biolabs, St. Paul, MN) were used in this study. Ten rats were treated in the first 48 h of life by a subcutaneous (s.c.) injection of 50 mg/kg eapsaicin (98%, lot no. 58F-7125; Sigma Chemical Co., St. Louis, MO) dissolved in saline vehicle containing 10% Tween 80 and 10% ethyl alcohol. Seven rats were also injected s.c. in the first 48 h of life with vehicle only. All animals were housed in a room maintained at a constant temperature of 22 *C on a 12/12 h light/dark cycle with food and water provided ad libitum. The groups are summarized in Table I.
Surgery All adult (16 weeks of age) rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (Nembutal, 45 mg/kg; Abbott Labs., IL) and the left femoral artery and vein were cannulated for the subsequent recording of arterial blood pressure and administration of drugs, respectively. The lines were externalized between the shoulder blades and all wounds were sutured and the margins were liberally coated with a local anesthetic ointment (Nupercainal, Ciba, Edison, NJ). The animals were allowed to recover from surgery for 3-7 days before experimental use (see below).
Nociceptive testing Nociceptive responses were determined using a TF apparatus as described previously33. Briefly, the apparatus consisted of a beam of focused radiant heat provided by a 50 W projector lamp. Radiant heat was focused systematically on the underside of the tail at one of 5 sites 8-10 mm apart with the most distal site approx. 4 cm from the tip. The TF latency was measured to the nearest 0.1 s as the time from onset of heating of the tall to withdrawal of the tail from the beam. A cut-off latency of 7 s was used to avoid tissue damage to the tail 39.
Experimental design Rats in the capsaiein-treated (n = 10) or vehicle-treated (n = 7) groups were given a single bolus dose of sodium pentobarbital (4.5-9.0 mg, i.v.) and allowed to recover to a light level of anesthesia (corneal and flexion withdrawal reflexes intact). In this comparatively light level of anesthesia, nociceptive withdrawal reflexes can be evoked without producing complex coordinated
escape behaviors39. The rheostat on the TF apparatus was adjusted such that at 7 s, the temperature as measured by a thermometer bulb was 65 *C. Baseline "IT lateneies were randomly recorded 3 times at each of 5 different sites approx. 8-10 mm apart for a total of 15 trials. The time between successive trials was 30 s. The rheostat was then adjusted such that at 7 s the temperature as measured by a thermometer bulb was 45 °C or 55 °C. The 15 trials were then repeated at each temperature as outlined above. The heat was then readjusted to 65 °C such that the baseline TF latency was usually between 1.8 and 2.4 s and 5-HT creatinine sulphate (Sigma Chemical Co., St. Louis, MO) was administered in an ascending dose-dependent manner (6, 12, 24, 48, 72, 96, 144 or 192/zg/kg, i.v.) until inhibition of the TF reflex to cutoff (7 s). All subsequent measurements of TF latency were made at the fastest rate of heating. Animals were tested 3, 33, 63 and 123 s following the i.v. administration of 5-HT. Latency measurements were continued at two-min intervals thereafter until the animal returned to the baseline latency established pre-drug. Arterial blood pressure and heart rate (HR) were monitored continuously throughout the experiments. The rat's behavioral reactions to each dose of i.v. 5-HT were assessed qualitatively as either present or absent 33. Two to three days following determination of the dose-response curve to i.v. 5-HT, the animals from each group were briefly anesthetized with ether, decapitated, the sacral spinal cord at the level of the iliac crest was severed with sharp scissors, and the spinal cords were extracted by hydraulic extrusion using a 16 gauge needle inserted into the spinal cord and flushed with 10 ml saline. The spinal cord was removed, wrapped in foil, and immediately frozen in liquid nitrogen. These samples were stored at -70 °C and subsequently a 2-cm piece of lumbar spinal cord, including the lumbar enlargement, was processed for sP and CGRP radioimmunoassay (RIA).
sP and CGRP RIA The content of sP and CGRP in each spinal cord was quantified by RIA. The lumbar enlargements of the frozen spinal cords were minced, homogenized and placed in boiling 0.1 N hydrochloric acid for 15 min and subsequently centrifuged at 12,000 g for 10 rain. Aliquots of the supernatant were. frozen for subsequent RIA. For sP, the samples were thawed, lyophilized and preincubated with antibody for 16 h at 4 *C. IlZLlabelled TyrS-sp containing 4% normal rabbit serum was added and incubated for an additional 24 h at 4 °C. The bound sP was separated from the free sP by the addition of a secondary goat anti-rabbit antibody. The final antibody concentration was 1:120,000 and the assay buffer consisted of 0.04 M KH2PO4, 1% BSA, 0.25% trasylol and 0.02% thimerosal, pH 6.5 with 4% PEG-8000 and 0.01 M EDTA. The absolute sensitivity of the assay was 10 pg/ml. For CGRP, the samples were preincubated for 16 h at 4 °C. I125-1abelled tyr°-CGRP (rat) was then added and incubated for an additional 16 h at 4 °C. Bound CGRP was separated from the free CGRP by the addition of a secondary donkey anti-goat antibody. The final antibody concentration was 1:84,000 and the assay buffer consisted of 0.06 M Na2HPO4, 0.013 M EDTA, 0.02% sodium azide, 0.1% triton-X, pH 7.4. The absolute sensitivity of the assay was 150 pg/ml. In either assay the inter- and intra-assay variation was less than 10%.
TABLE I
Data acquisition and statistical analyses
Summary of experimental groups Neonatal treatment
Number (n) Weight (g) Resting MAP (mmHg) Resting HR (b.p.m.) Baseline TF (s)
Capsaicin
Vehicle
10 482 + 12" 111 + 4 349 + 10 2.4 + 0.1
7 439 + 12 112 + 5 339 + 17 2.1 + 0.1
* Significantly different from vehicle-treated rats, P < 0.05.
All data are presented as means + S.E.M. The mean TF latencies are presented as raw data in seconds. Raw TF data were analyzed by sigmoidal curve fitting analysis which generated the ED5o and the gain of the 5-HT-mediated inhibition of the TF reflex. All other data were analyzed by analysis of variance with covariance (ANCOVA) 5°. Differences between individual means were determined by Student's modified t-test with a Bonferroni correction for multiple comparisons utilizing the modified error mean squared terms from the covariance analysis49. Correlations between inhibition of the TF reflex at the greatest doses of 5-HT used, and spinal contents of sP or CGRP were made with Spearman's rank correlation procedure for the capsaicin-treated rats. To standardize
214 comparisons, TF values were expressed as TF index (TFI) which is calculated as: (trial latency-baseline latency) divided by (cutoff latency-baseline latency) multiplied by 100 and expressed as a percentage depletion of p~ptide compared to the mean of vehicletreated rats.
7
0 neonatal vehicle • neonatal capsaicin
0
I
_/
I/
RESULTS
2 Comparison o f baseline T F latencies at different rates o f skin heating As the reported effects of neonatal capsaicin on heat-evoked nociceptive reflexes are inconsistent 4'7'n' 14,18-21,25,36,37, the rate of heating of the tail in this study was adjusted such that at cutoff latency (7 s) the temperature of the beam reached either 45, 55 or 65 °C. Therefore, any differences between the capsaicin-treated and vehicle-treated groups might readily become apparent. However, there was no difference in baseline TF latencies between capsaicin-treated rats and vehicletreated rats at any of the 3 rates of heating (Table II). Although, in both the capsaicin-treated and vehicletreated rats, there was a positive correlation between the rate of tail heating and TF latency; that is, TF latencies were significantly faster at the higher rate of heating. Effect o f i.v. 5 - H T TE 5-HT (6-96/~g/kg, i.v.) produced a dose-dependent inhibition of the TF reflex (n = 7) in vehicle-treated rats. The EDs0 (50% of maximal effect) for inhibition of the TF reflex was 48.1 + 11.3 /~g/kg. In contrast to vehicle-treated rats, 5-HT (6-192/~g/kg, i.v.) did not, as a group, significantly alter TF latency from baseline in capsaicin-treated rats (n = 10) (Fig. 1). In 6/10 capsaicintreated rats, 5-HT did not alter T F latency even at the greatest dose administered (192/~g/kg, i.v.); however, 3/10 of the capsaicin-treated rats exhibited slightly increased TF latencies at the greatest doses tested and 1/10 of the capsaicin-treated rats was completely inhibited to cut-off by 144 /,g/kg 5-HT. Baseline TF latency in capsaicin-treated rats was not significantly different from vehicle-treated rats (Table II). Cardiovascular. In both vehicle- (6-96 ~ug/kg, i.v.) and
TABLE II
~
~o
•-*-I ~
I
0
I B
, 3
10
, 30
100
300
dose 5-HT ( ~ g / k g )
Fig. 1. Alteration in the latency of the nociceptive tail-flick (TF) reflex produced by graded doses of intravenous (i.v.) serotonin (5-HT) in neonatal capsaicin-treated (0) (n = 10) or vehicle-treated rats (O) (n = 7). The dose administered is represented on the x-axis in pg/kg and changes in TF latency are represented on the y-axis in seconds. All data points are expressed as means + S.E.M.I.v. 5-HT produced a significant (P < 0.05) dose-dependent inhibition of the TF reflex (EDs0 = 48.1 + 11.3/~g/kg, gain = 22.3 + 7.5 s/log10dose; P < 0.05), whereas 5-HT administered to capsaicin-treated rats did not produce any significant change in TF latency (P > 0.05 for all comparisons).
capsaicin- (6-192 #g/kg, i.v,) treated rats, 5-HT typically produced a dose-related triphasic effect on arterial blood pressure and a bradycardia. The cardiovascular responses consisted of a brief, Bezold-Jarisch reflexly mediated bradycardia and associated decrease in arterial blood pressure followed by an increase in arterial blood
A
Neonatalvehicle
"~ 200]
DJ
rff Oj
B
T
T
T
30 sec
Neonatalcapsaicin
200]
5001
Summary of baseline TF latency at different rates of heating Neonatal treatment Vehicle Capsaicin a
Temperature at 7 s
Ou
45 °C
55 °C
65 °C
3.5 + 0.1 3.9 + 0.2
2.6 +_.0.1" 2.8 + 0.1"
2.1 + 0.1" 2.4 + 0.1"
a No difference between capsaiein- and vehicle-treated rats at all temperatures, P > 0.05. * Significantly different from 45 °C, P < 0.05.
T
12 pg/kg
T
4-8
pg/kg
T
72
pg/kg
Fig. 2. Illustration of typical pulsatile arterial blood pressure (PP; mmHg) and heart rate (HR; b.p.m.) responses to 5-HT (12, 48 or 72/~g/kg, i.v.) in (A) neonatal vehicle-treated rats or (B) neonatal capsaicin-treated rats. 5-HT was administered in the femoral vein. The time of the injection is indicated by the large arrow. Note the similarity in blood pressure and heart rate responses to 5-HT in neonatal vehicle- and capsaicin-treated rats.
215
A
0
•
vehicle n
-50 E
C
50 il
~
40
-100
",--" - 1 5 0 tic -200
~ 20 ~ lO
-250 -500
i
0
i
B
0
i
D
0
-10
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-10
-20 -20 E
-30
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< -40 ,
