Brain Research, 578 (1992) 186-196 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00.
186 BRES 17640
Bradykinin modulation of a spinal nociceptive reflex in the rat M.B. Bauer, Stephen T. Meller and G.E Gebhart Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, IA 52242 (USA) (Accepted 10 December 1991)
Key words: Pain; Tail-flick reflex; Visceral; Capsaicin; Vagus; Stellate ganglion; Nociception; Bradykinin; Visceral afferent
Bradykinin (BK) is a potent algesic compound. Therefore, we hypothesized that BK, acting as a peripheral noxious stimulus, would attenuate or inhibit responses to another noxious stimulus. When administered intravenously (i.v.) to rats lightly anesthesized with pentobarbital, BK produced a dose-dependent (12-144 pg/kg) inhibition of the nociceptive tail-flick (TF) reflex. BK also produced a dose-dependent decrease in mean arterial blood pressure, a subsequent increase in heart rate and an increase in the rate of respiration. The latency to the maximal effect of BK on the TF reflex was 10 s and was occasionally preceded by pseudoaffective responses at doses greater than 48 pg/kg BK. Neonatal treatment with capsaicin (50 mg/kg, subcutaneous) significantly attenuated BK-produced inhibition of the TF reflex indicating that BK was acting via peripheral afferents to inhibit the TF reflex. Reversible cold block or complete spinal transection at a low thoracic level (T9_12), but not reversible cold block at a high cervical level (C~_2), significantly attenuated BK-produced inhibition of the TF reflex, suggesting that BK activates afferents which enter the spinal cord between C2 and T 9. Pretreatment with intrathecally administered phentolamine (30 pg), an a-adrenoceptor antagonist, or methysergide (30/~g), a non-selective serotonin receptor antagonist, did not alter BKproduced inhibition of the TF reflex, further supporting the absence of activation of descending systems from the brainstem by i.v. BK. While coadministration of PGE 2 and BK significantly potentiated BK-produced inhibition of the TF reflex, neither bilateral removal of the steUate ganglion nor bilateral cervical vagotomy significantly affected the inhibitory action of i.v. BK on the TF reflex. These results suggest that i.v. BK inhibits the nociceptive TF reflex by activation of capsaicin-sensitive visceral afferents entering the spinal cord between C2 and T 9•
INTRODUCTION Bradykinin (BK) exerts various p o t e n t pharmacological effects, including smooth muscle contraction 4°, vasodilation 27'33, e d e m a 41 and pain 7'924. BK, normally
or intracoronary 51 administration have implicated B K in the etiology of cardiac pain. Additionally, elevated concentrations of BK have been observed in coronary sinus blood following acute occlusion of the left coronary artery 17,19.
present in the plasma in very low concentrations 6"19"26" 52, is considered to significantly contribute to the pain associated with acute tissue injury. I n d e e d , elevated plasma concentrations of B K are present during inflammation 42 and ischemia 17, conditions often associated with pain. E v e n in the absence of tissue injury, intradermal 8, intraarterial 7'9A5 or intraperitonea125'5° administration of
Recent reports d e m o n s t r a t e d that intravenous (i.v.) administration of a n o t h e r putative endogenous algesic chemical, serotonin, p r o d u c e d pseudaffective responses in rats and inhibited the nociceptive tail-flick reflex 28'29.
B K also produces pain and pseudaffective responses indicative of pain in h u m a n and n o n - h u m a n animals, respectively. B K has b e e n r e p o r t e d to directly stimulate A 6 - and C-fiber afferents from skin 3 as well as from muscle and viscera 12'22 and B K injected intraperitoneally p r o d u c e s objective and subjective reports of pain in humans 25, writhing responses in mice 53, and pseudoaffective responses in cats and dogs 1°. Intra-arterial injection of B K in dogs evokes pseudaffective responses 15. Responses e v o k e d by B K following epicardia143'51, intra-atrial a,5,51
present study was to characterize the algesic efficacy of i.v. B K in a similar m a n n e r and to investigate whether B K stimulates visceral afferents, particularly cardiopulm o n a r y afferents accessed from the right ('venous') side of the heart, following systemic administration, which m o r e closely reproduces natural situations. In this investigation, the effects of systemically administered B K and the neural pathways responsible for the B K - p r o d u c e d effects were investigated using a spinal nociceptive withdrawal reflex, the tail-flick (TF) reflex. A preliminary r e p o r t of some of this work has been presented 2.
In those studies, the nociceptive responses to i.v. serotonin were found to be m e d i a t e d by capsaicin-sensitive c a r d i o p u l m o n a r y vagal afferents 2s'29. The purpose of the
Correspondence: M.B. Bauer, Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA. Fax: (1) (319) 335-7903.
187 MATERIALS AND METHODS
Animals and surgery Sprague-Dawley rats (Biolab, St. Paul, MN) weighing 482 ± 9 g (S.E.M.) (232-591 g) were kept in a room maintained at 22°C on a 12/12 h light/dark cycle with food and water available ad libitum. Male rats were used in all experiments except those involving neonatal treatment with capsaicin in which both male and female rats were used. A total of 67 rats, divided into 11 groups (Table I), was used. All rats were initially anesthetized with an intraperitoneal injection of sodium pentobarbital (Nembutal, 45 mg/kg). The left femoral artery was cannulated for measurement of blood pressure and heart rate (HR), and the left femoral vein was cannulated for administration of BK. The venous and arterial catheters of rats in group 10 were tunneled subcutaneously and exteriorized at the back of the neck so that testing could be done 24-48 h after a low thoracic (T9_10) spinal cord transection. In all other rats, the right femoral vein was cannulated for continuous infusion of a 10% solution of sodium pentobarbital (50 mg/ml) to maintain a constant light level of anesthesia. For rats in group 10, a light state of anesthesia was maintained during the experiment by periodic injection of a 10% sodium pentobarbital solution into the venous line. All rats were tested while in a lightly anesthetized state. Wound margins were liberally coated with 1% dibucaine ointment (Nupercainal). The trachea of rats in groups 5, 6 and 11 was cannulated to insure adequate ventilation. Laminectomies were performed at C~_2 and T9_12 in rats from groups 5 and 9, respectively; crushed frozen normal saline was applied to the exposed spinal cord to reversibly block axonal conduction. During cervical spinal cord cold block (group 5), respiration ceased in four rats, and a respirator was used to artificially ventilate these rats (45-50 breaths/rain; 2.5-3.5 ml stroke volume). For rats in group 10, a laminectomy was performed at T9_12, and the exposed spinal cord was transected with a scalpel. Rats from this group were allowed to recover for 24-48 h before nociceptive testing. During the recovery period, bladders of these rats were expressed. Additional surgical manipulations were performed on animals in groups 6-8 and 11. In rats from groups 7 and 8, PE-10 tubing, 7 cm long, was inserted into the intrathecal space through a slit in the atlanto-occipital membrane to extend to the lumbar level of the spinal cord. After testing was completed, fast green dye was injected through the intrathecal catheter to mark the spinal cord level at which the drugs had been administered, and the spinal cords were hydraulically extruded. In rats from group 6, the cervical vagus nerve was exposed from the ventral side and cut above the level of the recurrent laryngeal nerve bilaterally. An axial approach was
TABLE I
Experimental groups Experimental group
n
Weight (g)a
Wt. range (g)
1 Latency to max. effect 2 Dosing interval 3 PGE 2 coadministration 4 Neonatal capsaicin/veh. 5 Cervical cold block 6 Bilateral vagotomy 7 Intrathecal methysergide 8 Intrathecal phentolamine 9 Thoracic cold block 10 Thoracic spinal transect. 11 Bilateral stellatectomy
5 5 7 10 9 7 4 4 3 3 10
541 494 511 384 451 499 493 502 526 501 476
± ± ± ± + ± ± + ± ± ±
10 13 12 38 32 16 3 5 13 7 12
519-571 467-543 467-563 232-551 396-526 459-591 461-537 450-540 494-547 477-514 393-555
Total
67
478 ±
9
232-591
a Mean + S.E.M.
used (on each side) for bilateral removal of the stellate ganglion from rats in group 11. Rats in group 4 were injected subcutaneously with capsaicin (50 mg/kg) or vehicle (80% saline/10% ethanol/10% Tween-80) within 48 h of birth. These ten rats (five capsaicin- and five vehicletreated) were used in the present experiments when 15-17 weeks of age (232-551 g).
Drugs Bradykinin acetate (Sigma Chemical Co., St. Louis, MO), dissolved in 0.9% saline to yield a solution with a concentration of 1 ~g/ml, was frozen in small aliquots for subsequent use. BK was administered i.v. in doses of 12, 24, 48, 72, 96 or 144/~g/kg followed by a 0.25 ml saline flush. I.v. administered drugs were injected over a 5-s period. The time to effect on TF latency and cardiovascular effects was measured from the end of the injection. At least three doses of BK were administered before and after surgical manipulations or pharmacological treatments. An injection of 0.25 ml vehicle (0.9% saline) was used as a control. Prostaglandin E~ (PGE2; Sigma Chemical Co.), dissolved in one part ethanol and nine parts 0.9% saline to yield a solution of 1 ~g/ml, was frozen in small aliquots for subsequent use. I.v. injections of PGE 2, with or without coadministration of BK, were followed by a 0.25 ml saline flush. Capsaicin (98%, Lot no. 58F-7125, Sigma Chemical Co.) was dissolved in a saline vehicle containing 10% ethanol and 10% Tween-80. Fresh solutions of methysergide maleate (Sandoz, East Hanover, N J), 3 ~g/~l, and phentolamine hydrochloride (CibaGeigy, Summit, N J), 3/~g//~l, were prepared in distilled water prior to each experiment. Ten/A of either drug solution followed by a 10 /A artificial cerebrospinal fluid flush were administered intrathecally 15 min before beginning the i.v. injections of BK and TF latency measurements. Nociceptive testing Rats were kept on a thermostatically controlled heating pad (36.5 _+ 0.5°C) and maintained in a lightly anesthetized state by a continuous i.v. infusion of a 10% pentobarbital solution during nociceptive testing (except group 10, which received periodic dosages of pentobarbital i.v. to maintain a light level of anesthesia). Nociceptive testing was begun after rats had recovered to a light level of anesthesia as determined and monitored by testing the corneal reflex, paw withdrawal to pinch, and TF withdrawal latency. A TF apparatus focused radiant heat (2 x 9 mm area) from a 50 W projector lamp to the underside of the rat's tail at one of five different sites which were 1 cm apart, beginning 3 cm from the distal end of the tail. TF latency was measured to the nearest 0.1 s by a photocell-timer circuit from the opening of a shutter until the rat removed its tail from the heat source. To establish the baseline TF latency, which was 2.0-2.5 s, TF latencies were measured 1 min apart at a minimum of three sites prior to initial administration of BK and before and after each surgical manipulation or pharmacological treatment. TF latencies after administration of BK were measured periodically until TF latency had returned to baseline. A cutoff latency of 7 s was used to avoid tissue damage to the tail 32. TF latency, arterial blood pressure, and HR were monitored through the entire experiment. Subsequent injections of BK were not made until TF latency, arterial blood pressure, and HR had returned to baseline values. Statistical analysis Baseline J T latencies are reported as the mean (s) + S.E.M. of three measurements determined prior to i.v. administration of BK. An effective dose-50 (EDso, the dose producing half of the maximal effect) was determined using sigmoidal curve fitting analysis23, and paired t-test was used to compare the pre- and post-treatment EDs0's within the same groups. Significance was defined as P < 0.05. All other data were compared using analysis of covariance (ANCOVA) and included a Bonferroni correction for multiple comparisons using the modified error mean square term from the ANCOVA 49.
188
12~/kg
RESULTS
z4 ~,.Q/kg
48t.~g/kg
. 200] I.v. BK produced a dose-dependent (12-144/~g/kg) inhibition of the TF reflex (Fig. 1A). The overall ED50 (determined by combining the pre-treatment data from groups 3, 5-9, and 11) was 44.7 + 3.2/~g/kg (n = 44). BK, administered up to a dose of 144/,g/kg, did not inhibit the TF reflex in four rats which were not included in any experiment. In the present experiments, at doses greater than 48 ag/kg BK, rats occasionally displayed pseudaffective responses such as closing of the eyes, flattening of the ears against the head, and running-like motion of the hindlimbs beginning approximately 5-6 s after injection of BK. BK produced a transient decrease in mean arterial blood pressure (MAP) and an increase in H R within 3 s of injection. Increasing the dosage of BK caused greater magnitude effects on MAP and H R between 12 and 48 /~g/kg. Greater doses of BK generally did not further affect MAP or HR. Typical examples from one rat are shown in Fig. 2. The rate of respiration, assessed visually, increased within 5 s after administration of BK and returned to its previous rate within approximately 30 s.
m~lO0 0J 500
]
I
mln
250
0 Fig. 2. Typical examples of the effects of i.v. BK on blood pressure (BP) and heart rate (HR). Within 3 s after the i.v. injection of BK, noted by the arrows. BP fell and HR subsequently increased. BP decreases and HR increases were dose-dependent between 12 and 48/,g/kg BK (Table II).
tenuation of the TF reflex by BK was maximal 10 s after injection in four rats and 15 s after injection in one rat. Because BK maximally affected the TF reflex 10 s after injection, measurements of TF latency in subsequent experiments were made 10 s after the end of the i.v. administration of BK.
Latency to maximal effect (group 1)
Dosing interval (group 2)
To determine when BK was acting to maximally inhibit the TF reflex, TF latencies were measured at different 5 s time points following injection of BK in separate trials. The effect of BK on the TF reflex was maximal when tested 10 s after the end of the i.v. injection of BK (24-48/*g/kg, n = 5; Fig. 1B). Only the TF latency measured 10 s following BK injection (5.6 + 0.8 s) was significantly greater than the baseline TF latency (1.9 + 0.2 s). The TF latency returned to baseline values 40 s after injection (2.3 + 0.1 s). Individually, at-
Because tachyphylaxis to a variety of responses evoked by BK has been reported in several systems 1"3'5'~4'31'4°' 43.45.51, the development of tachyphylaxis to BK-produced inhibition of the TF reflex following its i.v. administration was addressed in a second set of experiments. An initial dose-response curve was obtained by administering incrementing i.v. doses of BK 10 min apart. Thirty-five min after completion of the first dose-response curve, a second dose-response curve was obtained in the same rats by administering the same incrementing i.v. doses of BK 5 min apart (n = 5). The EDs0 for BK given at 10 min intervals, 57.7 + 14.4 pg/ kg, was not significantly different than the ED50 for BK given at 5 min intervals, 47.1 + 12.1 /~g/kg. Baseline MAP and H R were the same for both groups. Decreases in MAP and increases in H R produced by injections made 10 min apart were the same as those following injections made 5 min apart (Table II). To further examine the question of tachyphylaxis to i.v. BK, particularly regarding its effects on MAP and HR, a second 12 pg/kg dose of BK was given 10 min after determination of an initial dose-response curve to BK (12-96 pg/kg) in I1 rats. The mean TF latency after the first 12 pg/kg BK dose, 2.4 + 0.2 s, did not differ from that after the second 12 pg/kg dose, 2.6 + 0.2 s. In these same 11 rats, the mean decrease in MAP, 27 + 4 mmHg, and the mean increase in HR, 57 + 6 beats/min, produced by the initial 12 pg/kg BK dose did not differ
A "i7/ f61
B
71A +
C5 ~-4
_o 2
0
5 4 3 2
•
r" -', C10
O 30
100
Dose (p.g/kg)
300
•
. C
•
.
. O
. 10
. . . 20 30 40
' 50
60
Time (sec)
Fig. I. A: dose-dependent inhibition of the TF reflex by i.v. BK. The overall EDs0 was 44.7 + 3.2/~g/kg BK (n = 44). Log BK doses (ug/kg) tested are given on the x-axis, and TF latencies (s) on the y-axis. B: time to maximal inhibition of the TF reflex by i.v. BK (24-48 ~g/kg; n = 5). BK was administered i.v. over 5 s, the beginning of which is noted by the dotted line. The data in panels A and B above C represent baseline TF latency. Data reported as mean +_- S.E.M.
189 f r o m t h e m e a n c h a n g e s f o l l o w i n g t h e s e c o n d 12 /~g/kg
A
B K d o s e , 20 + 2 m m H g a n d 68 + 4 b e a t s / m i n , r e s p e c 7 5
v
> '4 o
_o 2
$
0 CI0
B 7
tively. T h e s e r e s u l t s d e m o n s t r a t e t h a t t a c h y p h y l a x i s d i d
J
%'6
n o t d e v e l o p t o t h e c a r d i o v a s c u l a r effects o f B K at a dosing i n t e r v a l o f 10 m i n . P G E 2 coadministration with B K (group 3)
•
bradykin|n
0
bradykinin + pGEz l
J
30
100
To d e t e r m i n e w h e t h e r P G E z, t h o u g h t t o s e n s i t i z e per i p h e r a l n o c i c e p t o r s x~'35'44'45, w o u l d p o t e n t i a t e t h e e f f e c t
--'3
300
o f B K o n t h e T F reflex, P G E 2 w a s c o a d m i n i s t e r e d w i t h B K . I.v. P G E 2 (0.25, 0.50 a n d 1 . 0 / ~ g / k g ) a l o n e d i d n o t
•
eapsaicin
~)
c h a n g e b a s e l i n e T F l a t e n c y . A t 0.25 a n d 0.50 p g / k g ,
.._.5
P G E 2 p r o d u c e d c h a n g e s in M A P a n d H R e q u i v a l e n t t o
~4
t h o s e p r o d u c e d b y c o n t r o l i n j e c t i o n s o f saline. A t 1.0
~3 -~2
/~g/kg, P G E 2 d i d n o t a l t e r M A P b u t a p p e a r e d t o slightly i n c r e a s e H R in s o m e rats.
1 0
C o a d m i n i s t r a t i o n o f P G E 2 (0.5 p g / k g ) w i t h B K ( 1 2 1 4 4 / ~ g / k g ) to t h e s a m e r a t s (15 m i n a f t e r t e s t i n g P G E 2 C lO
30
1DO 300
a l o n e ) significantly e n h a n c e d B K - p r o d u c e d i n h i b i t i o n o f
Dose (~g/kg)
t h e T F reflex in six o f s e v e n rats. A s s e e n in Fig. 3 A ,
Fig. 3. A: effect of co-administration of PGE 2 (0.5 pg/kg) with BK (12-144 pg/kg) on BK-produced inhibition of the TF reflex (n = 7). B: effect of neonatal capsaicin treatment on BK-produced inhibition of th,, TF reflex (n = 5/group). TF latencies (s) are reported on the ),-axes and log doses of BK (~g/kg) on the x-axes. Data above C represent baseline TF latency. Data reported as mean + S.E.M.
PGE 2 coadministration shifted the overall dose-response c u r v e t o t h e left, p r o d u c i n g a significant d e c r e a s e in t h e E D s 0 f r o m 55.7 + 7 . 4 p g / k g t o 30.9 _+ 4 . 5 / t g / k g . Baseline MAP
and HR were the same whether BK
was a d m i n i s t e r e d a l o n e o r w i t h P G E 2 , a l t h o u g h t h e d e c r e a s e in M A P f o l l o w i n g c o a d m i n i s t r a t i o n o f P G E 2 a n d
TABLE II BK-produced decreases in mean arterial pressure (MAP," mmHg) and increases in heart rate (HR; beats/min) Experimental group
Dosing intervals PGE 2 Coadministrat, Neonatal treatment Cervical cold block b Bilateral vagotomy Intrathecal methysergide Intrathecal phentolamine Thoracic cold block Thoracic transection Bilateral stellatectomy
Baseline M A P ~
I0 min: 5 min: pre: during: vehicle: capsaicin: pre: during: pre: post: pre: post: pre: post: pre: during: post:
118 110 112 115 116 100 125 53 113 135 92 100 90 88 81 97 92
+ + + + + + + + + + + + + + + + +
6 3 3 4 7 7* 4 2* 6 3* 9 2 3 3 6 5* 3
post: pre: post:
90 + 5 129 + 4 116 + 5
B K dose (pg/kg):
S.E. c
Baseline HR a
12
24
28
35 27 36 30 35 32 45 12 27 53 29 29 29 29 29 23 29
49 39 48 36* 50 43 49 13 41 63 35 33 33 37 29 26 36
58 48 57 52 55 39* 63 11 51 67 39 34 42 41 46 44 35
5 5 3 3 3 3 4 4 3 3 3 3 3 3 4 4 4
331 324 334 331 370 338 394 277 344 359 335 343 365 379 296 308 301
31 44 35
34 58 45
41 66 52*
9 3 3
a Data reported as mean + S.E.M. b Data reported for four rats with cessation of respiration during cold block. c Standard error of the mean calculated from (EMS/n) -vz. * Significantly different, P < 0.05.
B K dose (itg/kg):
S.E. C
12
24
48
28 34 18 20 29 34 11 20* 23 11 21 19 12 5 34 44 20
39 50 55 53 39 25 20 6 41 24 58 55 43 53 25 33 38
52 60 63 61 58 41 30 15 55 29* 66 84 50 56 28 45 40
63 61 62 59 71 92 43 53 70 45* 94 110 59 85 50 38 42
6 6 7 7 10 10 5 5 5 5 9 9 11 11 10 10 10
391 + 16 366 + 11 321 + 22*
44 30 30
68 46 42
70 51 43
4 6 6
+ + + + + + + + + + + + + + + + +
190
/A
B Cervical Cold Block
C Spinal Transection (T9_12)
Thoracic Cold Block
7
"G'e (D
6 5 4
~5 ~4
~3
,.
6 5
3 ]
0
•
• pro-0 during, rosp. ceased
186 BRES 17640
Bradykinin modulation of a spinal nociceptive reflex in the rat M.B. Bauer, Stephen T. Meller and G.E Gebhart Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, IA 52242 (USA) (Accepted 10 December 1991)
Key words: Pain; Tail-flick reflex; Visceral; Capsaicin; Vagus; Stellate ganglion; Nociception; Bradykinin; Visceral afferent
Bradykinin (BK) is a potent algesic compound. Therefore, we hypothesized that BK, acting as a peripheral noxious stimulus, would attenuate or inhibit responses to another noxious stimulus. When administered intravenously (i.v.) to rats lightly anesthesized with pentobarbital, BK produced a dose-dependent (12-144 pg/kg) inhibition of the nociceptive tail-flick (TF) reflex. BK also produced a dose-dependent decrease in mean arterial blood pressure, a subsequent increase in heart rate and an increase in the rate of respiration. The latency to the maximal effect of BK on the TF reflex was 10 s and was occasionally preceded by pseudoaffective responses at doses greater than 48 pg/kg BK. Neonatal treatment with capsaicin (50 mg/kg, subcutaneous) significantly attenuated BK-produced inhibition of the TF reflex indicating that BK was acting via peripheral afferents to inhibit the TF reflex. Reversible cold block or complete spinal transection at a low thoracic level (T9_12), but not reversible cold block at a high cervical level (C~_2), significantly attenuated BK-produced inhibition of the TF reflex, suggesting that BK activates afferents which enter the spinal cord between C2 and T 9. Pretreatment with intrathecally administered phentolamine (30 pg), an a-adrenoceptor antagonist, or methysergide (30/~g), a non-selective serotonin receptor antagonist, did not alter BKproduced inhibition of the TF reflex, further supporting the absence of activation of descending systems from the brainstem by i.v. BK. While coadministration of PGE 2 and BK significantly potentiated BK-produced inhibition of the TF reflex, neither bilateral removal of the steUate ganglion nor bilateral cervical vagotomy significantly affected the inhibitory action of i.v. BK on the TF reflex. These results suggest that i.v. BK inhibits the nociceptive TF reflex by activation of capsaicin-sensitive visceral afferents entering the spinal cord between C2 and T 9•
INTRODUCTION Bradykinin (BK) exerts various p o t e n t pharmacological effects, including smooth muscle contraction 4°, vasodilation 27'33, e d e m a 41 and pain 7'924. BK, normally
or intracoronary 51 administration have implicated B K in the etiology of cardiac pain. Additionally, elevated concentrations of BK have been observed in coronary sinus blood following acute occlusion of the left coronary artery 17,19.
present in the plasma in very low concentrations 6"19"26" 52, is considered to significantly contribute to the pain associated with acute tissue injury. I n d e e d , elevated plasma concentrations of B K are present during inflammation 42 and ischemia 17, conditions often associated with pain. E v e n in the absence of tissue injury, intradermal 8, intraarterial 7'9A5 or intraperitonea125'5° administration of
Recent reports d e m o n s t r a t e d that intravenous (i.v.) administration of a n o t h e r putative endogenous algesic chemical, serotonin, p r o d u c e d pseudaffective responses in rats and inhibited the nociceptive tail-flick reflex 28'29.
B K also produces pain and pseudaffective responses indicative of pain in h u m a n and n o n - h u m a n animals, respectively. B K has b e e n r e p o r t e d to directly stimulate A 6 - and C-fiber afferents from skin 3 as well as from muscle and viscera 12'22 and B K injected intraperitoneally p r o d u c e s objective and subjective reports of pain in humans 25, writhing responses in mice 53, and pseudoaffective responses in cats and dogs 1°. Intra-arterial injection of B K in dogs evokes pseudaffective responses 15. Responses e v o k e d by B K following epicardia143'51, intra-atrial a,5,51
present study was to characterize the algesic efficacy of i.v. B K in a similar m a n n e r and to investigate whether B K stimulates visceral afferents, particularly cardiopulm o n a r y afferents accessed from the right ('venous') side of the heart, following systemic administration, which m o r e closely reproduces natural situations. In this investigation, the effects of systemically administered B K and the neural pathways responsible for the B K - p r o d u c e d effects were investigated using a spinal nociceptive withdrawal reflex, the tail-flick (TF) reflex. A preliminary r e p o r t of some of this work has been presented 2.
In those studies, the nociceptive responses to i.v. serotonin were found to be m e d i a t e d by capsaicin-sensitive c a r d i o p u l m o n a r y vagal afferents 2s'29. The purpose of the
Correspondence: M.B. Bauer, Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA. Fax: (1) (319) 335-7903.
187 MATERIALS AND METHODS
Animals and surgery Sprague-Dawley rats (Biolab, St. Paul, MN) weighing 482 ± 9 g (S.E.M.) (232-591 g) were kept in a room maintained at 22°C on a 12/12 h light/dark cycle with food and water available ad libitum. Male rats were used in all experiments except those involving neonatal treatment with capsaicin in which both male and female rats were used. A total of 67 rats, divided into 11 groups (Table I), was used. All rats were initially anesthetized with an intraperitoneal injection of sodium pentobarbital (Nembutal, 45 mg/kg). The left femoral artery was cannulated for measurement of blood pressure and heart rate (HR), and the left femoral vein was cannulated for administration of BK. The venous and arterial catheters of rats in group 10 were tunneled subcutaneously and exteriorized at the back of the neck so that testing could be done 24-48 h after a low thoracic (T9_10) spinal cord transection. In all other rats, the right femoral vein was cannulated for continuous infusion of a 10% solution of sodium pentobarbital (50 mg/ml) to maintain a constant light level of anesthesia. For rats in group 10, a light state of anesthesia was maintained during the experiment by periodic injection of a 10% sodium pentobarbital solution into the venous line. All rats were tested while in a lightly anesthetized state. Wound margins were liberally coated with 1% dibucaine ointment (Nupercainal). The trachea of rats in groups 5, 6 and 11 was cannulated to insure adequate ventilation. Laminectomies were performed at C~_2 and T9_12 in rats from groups 5 and 9, respectively; crushed frozen normal saline was applied to the exposed spinal cord to reversibly block axonal conduction. During cervical spinal cord cold block (group 5), respiration ceased in four rats, and a respirator was used to artificially ventilate these rats (45-50 breaths/rain; 2.5-3.5 ml stroke volume). For rats in group 10, a laminectomy was performed at T9_12, and the exposed spinal cord was transected with a scalpel. Rats from this group were allowed to recover for 24-48 h before nociceptive testing. During the recovery period, bladders of these rats were expressed. Additional surgical manipulations were performed on animals in groups 6-8 and 11. In rats from groups 7 and 8, PE-10 tubing, 7 cm long, was inserted into the intrathecal space through a slit in the atlanto-occipital membrane to extend to the lumbar level of the spinal cord. After testing was completed, fast green dye was injected through the intrathecal catheter to mark the spinal cord level at which the drugs had been administered, and the spinal cords were hydraulically extruded. In rats from group 6, the cervical vagus nerve was exposed from the ventral side and cut above the level of the recurrent laryngeal nerve bilaterally. An axial approach was
TABLE I
Experimental groups Experimental group
n
Weight (g)a
Wt. range (g)
1 Latency to max. effect 2 Dosing interval 3 PGE 2 coadministration 4 Neonatal capsaicin/veh. 5 Cervical cold block 6 Bilateral vagotomy 7 Intrathecal methysergide 8 Intrathecal phentolamine 9 Thoracic cold block 10 Thoracic spinal transect. 11 Bilateral stellatectomy
5 5 7 10 9 7 4 4 3 3 10
541 494 511 384 451 499 493 502 526 501 476
± ± ± ± + ± ± + ± ± ±
10 13 12 38 32 16 3 5 13 7 12
519-571 467-543 467-563 232-551 396-526 459-591 461-537 450-540 494-547 477-514 393-555
Total
67
478 ±
9
232-591
a Mean + S.E.M.
used (on each side) for bilateral removal of the stellate ganglion from rats in group 11. Rats in group 4 were injected subcutaneously with capsaicin (50 mg/kg) or vehicle (80% saline/10% ethanol/10% Tween-80) within 48 h of birth. These ten rats (five capsaicin- and five vehicletreated) were used in the present experiments when 15-17 weeks of age (232-551 g).
Drugs Bradykinin acetate (Sigma Chemical Co., St. Louis, MO), dissolved in 0.9% saline to yield a solution with a concentration of 1 ~g/ml, was frozen in small aliquots for subsequent use. BK was administered i.v. in doses of 12, 24, 48, 72, 96 or 144/~g/kg followed by a 0.25 ml saline flush. I.v. administered drugs were injected over a 5-s period. The time to effect on TF latency and cardiovascular effects was measured from the end of the injection. At least three doses of BK were administered before and after surgical manipulations or pharmacological treatments. An injection of 0.25 ml vehicle (0.9% saline) was used as a control. Prostaglandin E~ (PGE2; Sigma Chemical Co.), dissolved in one part ethanol and nine parts 0.9% saline to yield a solution of 1 ~g/ml, was frozen in small aliquots for subsequent use. I.v. injections of PGE 2, with or without coadministration of BK, were followed by a 0.25 ml saline flush. Capsaicin (98%, Lot no. 58F-7125, Sigma Chemical Co.) was dissolved in a saline vehicle containing 10% ethanol and 10% Tween-80. Fresh solutions of methysergide maleate (Sandoz, East Hanover, N J), 3 ~g/~l, and phentolamine hydrochloride (CibaGeigy, Summit, N J), 3/~g//~l, were prepared in distilled water prior to each experiment. Ten/A of either drug solution followed by a 10 /A artificial cerebrospinal fluid flush were administered intrathecally 15 min before beginning the i.v. injections of BK and TF latency measurements. Nociceptive testing Rats were kept on a thermostatically controlled heating pad (36.5 _+ 0.5°C) and maintained in a lightly anesthetized state by a continuous i.v. infusion of a 10% pentobarbital solution during nociceptive testing (except group 10, which received periodic dosages of pentobarbital i.v. to maintain a light level of anesthesia). Nociceptive testing was begun after rats had recovered to a light level of anesthesia as determined and monitored by testing the corneal reflex, paw withdrawal to pinch, and TF withdrawal latency. A TF apparatus focused radiant heat (2 x 9 mm area) from a 50 W projector lamp to the underside of the rat's tail at one of five different sites which were 1 cm apart, beginning 3 cm from the distal end of the tail. TF latency was measured to the nearest 0.1 s by a photocell-timer circuit from the opening of a shutter until the rat removed its tail from the heat source. To establish the baseline TF latency, which was 2.0-2.5 s, TF latencies were measured 1 min apart at a minimum of three sites prior to initial administration of BK and before and after each surgical manipulation or pharmacological treatment. TF latencies after administration of BK were measured periodically until TF latency had returned to baseline. A cutoff latency of 7 s was used to avoid tissue damage to the tail 32. TF latency, arterial blood pressure, and HR were monitored through the entire experiment. Subsequent injections of BK were not made until TF latency, arterial blood pressure, and HR had returned to baseline values. Statistical analysis Baseline J T latencies are reported as the mean (s) + S.E.M. of three measurements determined prior to i.v. administration of BK. An effective dose-50 (EDso, the dose producing half of the maximal effect) was determined using sigmoidal curve fitting analysis23, and paired t-test was used to compare the pre- and post-treatment EDs0's within the same groups. Significance was defined as P < 0.05. All other data were compared using analysis of covariance (ANCOVA) and included a Bonferroni correction for multiple comparisons using the modified error mean square term from the ANCOVA 49.
188
12~/kg
RESULTS
z4 ~,.Q/kg
48t.~g/kg
. 200] I.v. BK produced a dose-dependent (12-144/~g/kg) inhibition of the TF reflex (Fig. 1A). The overall ED50 (determined by combining the pre-treatment data from groups 3, 5-9, and 11) was 44.7 + 3.2/~g/kg (n = 44). BK, administered up to a dose of 144/,g/kg, did not inhibit the TF reflex in four rats which were not included in any experiment. In the present experiments, at doses greater than 48 ag/kg BK, rats occasionally displayed pseudaffective responses such as closing of the eyes, flattening of the ears against the head, and running-like motion of the hindlimbs beginning approximately 5-6 s after injection of BK. BK produced a transient decrease in mean arterial blood pressure (MAP) and an increase in H R within 3 s of injection. Increasing the dosage of BK caused greater magnitude effects on MAP and H R between 12 and 48 /~g/kg. Greater doses of BK generally did not further affect MAP or HR. Typical examples from one rat are shown in Fig. 2. The rate of respiration, assessed visually, increased within 5 s after administration of BK and returned to its previous rate within approximately 30 s.
m~lO0 0J 500
]
I
mln
250
0 Fig. 2. Typical examples of the effects of i.v. BK on blood pressure (BP) and heart rate (HR). Within 3 s after the i.v. injection of BK, noted by the arrows. BP fell and HR subsequently increased. BP decreases and HR increases were dose-dependent between 12 and 48/,g/kg BK (Table II).
tenuation of the TF reflex by BK was maximal 10 s after injection in four rats and 15 s after injection in one rat. Because BK maximally affected the TF reflex 10 s after injection, measurements of TF latency in subsequent experiments were made 10 s after the end of the i.v. administration of BK.
Latency to maximal effect (group 1)
Dosing interval (group 2)
To determine when BK was acting to maximally inhibit the TF reflex, TF latencies were measured at different 5 s time points following injection of BK in separate trials. The effect of BK on the TF reflex was maximal when tested 10 s after the end of the i.v. injection of BK (24-48/*g/kg, n = 5; Fig. 1B). Only the TF latency measured 10 s following BK injection (5.6 + 0.8 s) was significantly greater than the baseline TF latency (1.9 + 0.2 s). The TF latency returned to baseline values 40 s after injection (2.3 + 0.1 s). Individually, at-
Because tachyphylaxis to a variety of responses evoked by BK has been reported in several systems 1"3'5'~4'31'4°' 43.45.51, the development of tachyphylaxis to BK-produced inhibition of the TF reflex following its i.v. administration was addressed in a second set of experiments. An initial dose-response curve was obtained by administering incrementing i.v. doses of BK 10 min apart. Thirty-five min after completion of the first dose-response curve, a second dose-response curve was obtained in the same rats by administering the same incrementing i.v. doses of BK 5 min apart (n = 5). The EDs0 for BK given at 10 min intervals, 57.7 + 14.4 pg/ kg, was not significantly different than the ED50 for BK given at 5 min intervals, 47.1 + 12.1 /~g/kg. Baseline MAP and H R were the same for both groups. Decreases in MAP and increases in H R produced by injections made 10 min apart were the same as those following injections made 5 min apart (Table II). To further examine the question of tachyphylaxis to i.v. BK, particularly regarding its effects on MAP and HR, a second 12 pg/kg dose of BK was given 10 min after determination of an initial dose-response curve to BK (12-96 pg/kg) in I1 rats. The mean TF latency after the first 12 pg/kg BK dose, 2.4 + 0.2 s, did not differ from that after the second 12 pg/kg dose, 2.6 + 0.2 s. In these same 11 rats, the mean decrease in MAP, 27 + 4 mmHg, and the mean increase in HR, 57 + 6 beats/min, produced by the initial 12 pg/kg BK dose did not differ
A "i7/ f61
B
71A +
C5 ~-4
_o 2
0
5 4 3 2
•
r" -', C10
O 30
100
Dose (p.g/kg)
300
•
. C
•
.
. O
. 10
. . . 20 30 40
' 50
60
Time (sec)
Fig. I. A: dose-dependent inhibition of the TF reflex by i.v. BK. The overall EDs0 was 44.7 + 3.2/~g/kg BK (n = 44). Log BK doses (ug/kg) tested are given on the x-axis, and TF latencies (s) on the y-axis. B: time to maximal inhibition of the TF reflex by i.v. BK (24-48 ~g/kg; n = 5). BK was administered i.v. over 5 s, the beginning of which is noted by the dotted line. The data in panels A and B above C represent baseline TF latency. Data reported as mean +_- S.E.M.
189 f r o m t h e m e a n c h a n g e s f o l l o w i n g t h e s e c o n d 12 /~g/kg
A
B K d o s e , 20 + 2 m m H g a n d 68 + 4 b e a t s / m i n , r e s p e c 7 5
v
> '4 o
_o 2
$
0 CI0
B 7
tively. T h e s e r e s u l t s d e m o n s t r a t e t h a t t a c h y p h y l a x i s d i d
J
%'6
n o t d e v e l o p t o t h e c a r d i o v a s c u l a r effects o f B K at a dosing i n t e r v a l o f 10 m i n . P G E 2 coadministration with B K (group 3)
•
bradykin|n
0
bradykinin + pGEz l
J
30
100
To d e t e r m i n e w h e t h e r P G E z, t h o u g h t t o s e n s i t i z e per i p h e r a l n o c i c e p t o r s x~'35'44'45, w o u l d p o t e n t i a t e t h e e f f e c t
--'3
300
o f B K o n t h e T F reflex, P G E 2 w a s c o a d m i n i s t e r e d w i t h B K . I.v. P G E 2 (0.25, 0.50 a n d 1 . 0 / ~ g / k g ) a l o n e d i d n o t
•
eapsaicin
~)
c h a n g e b a s e l i n e T F l a t e n c y . A t 0.25 a n d 0.50 p g / k g ,
.._.5
P G E 2 p r o d u c e d c h a n g e s in M A P a n d H R e q u i v a l e n t t o
~4
t h o s e p r o d u c e d b y c o n t r o l i n j e c t i o n s o f saline. A t 1.0
~3 -~2
/~g/kg, P G E 2 d i d n o t a l t e r M A P b u t a p p e a r e d t o slightly i n c r e a s e H R in s o m e rats.
1 0
C o a d m i n i s t r a t i o n o f P G E 2 (0.5 p g / k g ) w i t h B K ( 1 2 1 4 4 / ~ g / k g ) to t h e s a m e r a t s (15 m i n a f t e r t e s t i n g P G E 2 C lO
30
1DO 300
a l o n e ) significantly e n h a n c e d B K - p r o d u c e d i n h i b i t i o n o f
Dose (~g/kg)
t h e T F reflex in six o f s e v e n rats. A s s e e n in Fig. 3 A ,
Fig. 3. A: effect of co-administration of PGE 2 (0.5 pg/kg) with BK (12-144 pg/kg) on BK-produced inhibition of the TF reflex (n = 7). B: effect of neonatal capsaicin treatment on BK-produced inhibition of th,, TF reflex (n = 5/group). TF latencies (s) are reported on the ),-axes and log doses of BK (~g/kg) on the x-axes. Data above C represent baseline TF latency. Data reported as mean + S.E.M.
PGE 2 coadministration shifted the overall dose-response c u r v e t o t h e left, p r o d u c i n g a significant d e c r e a s e in t h e E D s 0 f r o m 55.7 + 7 . 4 p g / k g t o 30.9 _+ 4 . 5 / t g / k g . Baseline MAP
and HR were the same whether BK
was a d m i n i s t e r e d a l o n e o r w i t h P G E 2 , a l t h o u g h t h e d e c r e a s e in M A P f o l l o w i n g c o a d m i n i s t r a t i o n o f P G E 2 a n d
TABLE II BK-produced decreases in mean arterial pressure (MAP," mmHg) and increases in heart rate (HR; beats/min) Experimental group
Dosing intervals PGE 2 Coadministrat, Neonatal treatment Cervical cold block b Bilateral vagotomy Intrathecal methysergide Intrathecal phentolamine Thoracic cold block Thoracic transection Bilateral stellatectomy
Baseline M A P ~
I0 min: 5 min: pre: during: vehicle: capsaicin: pre: during: pre: post: pre: post: pre: post: pre: during: post:
118 110 112 115 116 100 125 53 113 135 92 100 90 88 81 97 92
+ + + + + + + + + + + + + + + + +
6 3 3 4 7 7* 4 2* 6 3* 9 2 3 3 6 5* 3
post: pre: post:
90 + 5 129 + 4 116 + 5
B K dose (pg/kg):
S.E. c
Baseline HR a
12
24
28
35 27 36 30 35 32 45 12 27 53 29 29 29 29 29 23 29
49 39 48 36* 50 43 49 13 41 63 35 33 33 37 29 26 36
58 48 57 52 55 39* 63 11 51 67 39 34 42 41 46 44 35
5 5 3 3 3 3 4 4 3 3 3 3 3 3 4 4 4
331 324 334 331 370 338 394 277 344 359 335 343 365 379 296 308 301
31 44 35
34 58 45
41 66 52*
9 3 3
a Data reported as mean + S.E.M. b Data reported for four rats with cessation of respiration during cold block. c Standard error of the mean calculated from (EMS/n) -vz. * Significantly different, P < 0.05.
B K dose (itg/kg):
S.E. C
12
24
48
28 34 18 20 29 34 11 20* 23 11 21 19 12 5 34 44 20
39 50 55 53 39 25 20 6 41 24 58 55 43 53 25 33 38
52 60 63 61 58 41 30 15 55 29* 66 84 50 56 28 45 40
63 61 62 59 71 92 43 53 70 45* 94 110 59 85 50 38 42
6 6 7 7 10 10 5 5 5 5 9 9 11 11 10 10 10
391 + 16 366 + 11 321 + 22*
44 30 30
68 46 42
70 51 43
4 6 6
+ + + + + + + + + + + + + + + + +
190
/A
B Cervical Cold Block
C Spinal Transection (T9_12)
Thoracic Cold Block
7
"G'e (D
6 5 4
~5 ~4
~3
,.
6 5
3 ]
0
•
• pro-0 during, rosp. ceased
