European Journal of Pharmacology, 45 (1977) 13--22 © Elsevier/North-Holland Biomedical Press
13
C H A N G E S IN R A T H I N D L I M B V A S C U L A R R E S I S T A N C E F O L L O W I N G INTRACEREBROVENTRICULAR DRUG ADMINISTRATION JOHN M. ELLIOTT and DAVID W.J. CLARK Department of Pharmacology, Otago Medical School, Dunedin, New Zealand
Received 8 February 1977, revised MS received 13 April 1977, accepted 17 May 1977
J.M. ELLIOTT and D.W.J. CLARK, Changes in rat hind limb vascular resistance following intracerebroventricular drug administration, European J. Pharmacol. 45 (1977) 13--22. The right hind limbs of rats (which had previously implanted intraventricular guide cannulae) were isolated from the systemic circulation, but with the nerves to the limb remaining intact, and perfused using a constant output blood pump. Using this preparation, changes in vascular resistance, blood pressure and heart rate were monitored following injection of noradrenaline, phentolamine and propranolol into the lateral cerebral ventricles (i.c.v.) of rats anaesthetised with a-chloralose. All three drugs lowered blood pressure. Noradrenaline administered i.c.v, induced a nervously mediated vasodilatation and an insignificant fall in heart rate whereas i.c.v, phentolamine administration was followed by a nervously mediated vasoconstriction in the isolated hind limb and a gradual rise in heart rate. After i.c.v, administration of propranolol there was no evidence of an immediate nervously mediated vasodilatation but heart rate fell significantly. Following i.c.v, phentolamine or propranolol vasodilatation did not occur in the hind limb until after the time taken for circulating blood ro reach the isolated vascular bed. The vasodilatatory and hypotensive responses to i.c.v, noradrenaline were not evident following prior i.c.v, injection of phentolamine. These results indicate the suitability of this preparation for investigations of central actions of other drugs. Antihypertensive agents Perfusion pressure
I.c.v. injections Heart rate
Blood pressure Noradrenaline
1. I n t r o d u c t i o n Several drugs have r e c e n t l y b e e n introduced into antihypertensive therapy which are t h o u g h t to e x e r t h y p o t e n s i v e actions b y w a y o f t h e central n e r v o u s s y s t e m . Such drugs i n c l u d e reserpine, a - m e t h y l d o p a , c l o n i d i n e , i n d o r a m i n and f l - a d r e n o r e c e p t o r b l o c k i n g drugs (Laverty, 1 9 7 3 ; van Zwieten, 1 9 7 3 ; D a y and R o c h , 1 9 7 4 ; B a u m and S h r o p s h i r e , 1 9 7 5 ; Scriabine et al., 1 9 7 6 ) . The investigation o f these drugs has p r o m p t e d intensive research i n t o the n e u r o p h y s i o l o g i c a l basis o f the central c o n t r o l o f cardiovascular f u n c t i o n i n g . The greatest b o d y o f evidence so far p r e s e n t e d is t h a t i m p l i c a t i n g e n d o g e n o u s n o r a d r e n a l i n e in the central c o n t r o l o f arterial b l o o d pressure
Phentolamine
Propranolol
(Day and R o a c h , 1974; Chalmers, 1 9 7 5 ; Haeusler, 1975). In m o s t earlier investigations, b l o o d pressure and h e a r t rate have b e e n m o n i t o r e d following the injection o f n o r a d r e n a l i n e into the cerebral v e n t r i c u l a r s y s t e m . Generally such a d m i n i s t r a t i o n has i n d u c e d a fall in b o t h b l o o d pressure and h e a r t rate. In r e c e n t years s o m e researchers have m o n i t o r e d electrical activity in a s e c t i o n e d s p l a n c h n i c nerve ( S c h m i t t and S c h m i t t , 1 9 6 9 ; B a u m and Shropshire, 1 9 7 0 , ] 9 7 3 ; Pichler and Kobinger, 1 9 7 6 ) . In this w a y t h e y have estim a t e d general changes in s y m p a t h e t i c o u t f l o w f r o m the central n e r v o u s s y s t e m . These studies have s h o w n t h a t c e n t r a l l y a d m i n i s t e r e d n o r a d r e n a l i n e decreases i m p u l s e flow along
14
the sectioned sympathetic nerve trunk. However, such measurement of electrical activity is not specific for impulse flow which is destined to supply the walls of blood vessels. Use of the innervated but vascularly isolated rat hind limb preparation {Laverty, 1962), perfused with blood at a constant rate, enables observation of vascular end organ responses to alteration in autonomic nervous activity induced by central administration of drugs. Furthermore, the incorporation of a delay coil into the perfusion circuit enables clear separation of nervously mediated and humorally mediated changes in vascular resistance. This paper describes response of perfusion pressure (proportional to the vascular resistance of the hind limb vascular bed), blood pressure and heart rate to centrally administered noradrenaline, phentolamine and propranolol. A preliminary report of some of the work has been given to the Otago Medical School Research Society (Elliott and Clark, 1976). 2. Materials and methods
J.M. E L L I O T T , D.W.J. C L A R K
2.2. The isolated hind limb preparation The left femoral vein was cannulated under ether anaesthesia, anaesthesia was maintained with a-chloralose {80 mg/kg i.v.) and the animal's trachea cannulated. The right hind limb was isolated from the main circulation (Laverty, 1962) but the femoral and sciatic nerves and the femoral vein were left intact. The preparation was perfused with blood, at the rate of 1 ml/min, by means of a non-haemolysing, constant o u t p u t blood pump constructed in the workshops of the Wellcome Medical Research Institute, Dunedin, N.Z. This pump and the tubing making up the perfusion circuit were primed with blood from a donor rat. A delay coil in the perfusion circuit ensured that blood reached the isolated hind limb between 6 and 7 min after being drawn from the opposite femoral artery. Blood pressure and perfusion pressure (proportional to vascular resistance) were monitored using pressure transducers and a pen recorder. Heart rates were determined from the ECG.
2.1. Implantation o f i.c.v, guide cannulae
2.3. Experimental procecure
Male Wistar rats (250--350 g) were used. Each rat was anaesthetised with sodium pentobarbitone (20 mg/kg i.p.) and ketamine (100 mg/kg i.p. injected contralaterally 15 min later) and placed in a stereotaxic apparatus. The skull surface was exposed and cleared, and a hole was drilled through the left parietal bone to position an anchoring screw. A second hole was then drilled 1 mm lateral and caudal to the bregma and a 5 mm guide cannula, similar to that described by Hayden et al. {1966) was lowered through this hole so that its bevelled tip came to lie within the right lateral ventricle. The perspex section of the cannula was then cemented to the anchoring screw and to the skull surface. The wound was sutured and the rat allowed to recover for at least one week before being used for perfusion experiments.
2.3.1. Test for nerve function As a test for the integrity of the autonomic nerve supply to the hind limb, i.v. noradrenaline (0.5 pg/kg) was injected at the beginning of every experiment and responses of blood pressure, perfusion pressure and heart rate were observed. 2.3.2. I.c.v. injections A length of 27 gauge needle was inserted into a short length of polyethylene tubing, the other end of which was attached to a 50 pl Hamilton syringe. The syringe, tubing and needle were then filled with the solution to be injected. The 27 gauge needle was then inserted into the chronically implanted guide cannula to a depth of 1 cm so that its tip was at the tip of the metal section of the guide cannula. In this way drugs could be injected
HINDLIMB V A S C U L A R BED RESPONSES TO ICV DRUG ADMINISTRATION
directly into the cerebral ventricle. Preliminary experiments were performed to find the minimum dose of noradrenaline which when administered i.c.v, produced an effect on hind limb perfusion pressure. After a stable perfusion pressure had been recorded for at least 10 min, 10 #1 of either noradrenaline (1 #g in 0.9% NaC1) or 0.9% NaC1 was injected i.c.v, at intervals of 30 min. Responses of perfusion pressure, blood pressure and heart rate to 2 i.c.v, injections of noradrenaline were recorded in 5 rats. In another 3 rats responses to 1 i.c.v, injection of noradrenaline were recorded. These 8 rats are referred to as "control rats" in the Results section. In 3 of the above preparations, and in a further 7 rats which had received no other centrally administered drug, phentolamine (100 pg) was injected i.c.v. Responses of perfusion pressure, blood pressure and heart rate were recorded. When perfusion pressure had again stabilized (10--15 min after the injection of phentolamine), 0.9% N a C 1 (10 pl) was injected to ensure no phentolamine remained in the guide cannula. At least 10 min later noradrenaline (1 pg in 10 pl 0.9% NaC1) was injected i.c.v, in the first group of 3 rats and in 2 further rats from the second group, and the responses o f perfusion pressure, blood pressure and heart rate were monitored. In a further 7 rats which had received no other centrally administered drug, propranolol (100 pg in 10 pl 0.9% NaC1) was injected i.c.v. Responses of perfusion pressure, blood pressure and heart rate were recorded. At least 60 min after i.c.v, injection of propranolol the femoral and sciatic nerves supplying the vascularly isolated hind limbs were sectioned and the resulting fall in perfusion pressure recorded.
2.3.3. Effects o f i.a. noradrenaline The influence of i.c.v, phentolamine on the pressor effectiveness of i.a. noradrenaline in the isolated hind limb was investigated in 6 preparations. Prior to i.c.v, injection of phentolamine, 0.1 pg noradrenaline (20 pl of 5
15
pg/ml in 0.9% NaC1 solution) was injected i.a. 2--3 times and perfusion pressure responses were averaged. At least 12 min after phentolamine (100 pg i.c.v.) two further doses of noradrenaline (0.1 pg i.a.) were given. Responses were again averaged and the pre- and post-phentolamine responses compared by the paired t-test.
2.3.4. Atropinised hind limb In 1 further experiment acetylcholine (0.1 pg in 100 pl) was injected into the hind limb (i.a.). After 10 min, atropine (1 mg in 100 pl) was injected i.a., followed by acetylcholine (0.1 pg i.a.). 5 min later, noradrenaline (1 #g i.c.v.) was administered and responses were recorded for 7 min, at which time a further dose of acetylcholine (0.1 pg i.a.) was injected. 2.3.5. I.c.v. indian ink At the completion of every experiment, indian ink (10 pl) was injected i.c.v. The brain was immediately removed from the skull and sectioned for examination of the distribution of the ink. 2. 4. Drugs The following drugs were administered: atropine sulphate, MacFarlan Smith Ltd.; noradrenaline bitartrate (Levophed), Winthrop; phentolamine mesylate (Rogitine), Ciba; d,l-propranolol hydrochloride, Sigma.
3. Results
3.1. Results o f distribution o f iophendylate and indian ink X-ray examination confirmed that the right lateral ventricle had been accurately cannulated. The radio-opaque iophendylate outlined the semicircular shaped lateral ventricles and the third ventricle below. The bony structures of the ear masked the hind brain regions and so the disposition of the iophendylate
16
J.M. ELLIOTT, D.W.J. CLARK
c o u l d n o t be f u r t h e r d e t e r m i n e d . Sectioning o f the brain to e x a m i n e the dist r i b u t i o n o f i.c.v, indian ink always s h o w e d the d y e t o be present in b o t h lateral ventricles and as far caudal as the f o u r t h ventricle and cisterna magna. Usually some indian ink had diffused into the s u b a r a c h n o i d space a r o u n d the spinal c o r d at the level o f C2 and C3.
3.2. Response to i.v. noradrenaline I.v. n o r a d r e n a l i n e (0.5 pg/kg), injected at the beginning o f e v e r y e x p e r i m e n t , i n d u c e d a rise in b l o o d pressure averaging 50 m m Hg. This pressor r e s p o n s e was a c c o m p a n i e d b y a (reflex) fall in p e r f u s i o n pressure o f 1 5 - - 3 0 m m Hg in the vascularly isolated i n n e r v a t e d hind limb and a fall in h e a r t rate (fig. 1).
3.3. Responses to i.c.v, noradrenaline Sections o f tracing o b t a i n e d d u r i n g an e x p e r i m e n t are p r e s e n t e d in fig. 2. Fig. 3 shows a c o m p a r i s o n o f the m e a n responses o f
'8°F 160F 140 F
120F I00L-
160F
2E I
I
I
I
I
I
I
I
I
Fig. 1. Arterial blood pressure (upper level, mm Hg), perfusion pressure (middle level, mm Hg) and heart
rate (lower level, beats/min) responses of an animal to 0.5 ~g/kg i.v. noradrenaline (NA, at arrows). The regular transient falls in perfusion pressure and increases in arterial blood pressure are a pump artefact. Heart rate was recorded at the points indicated. Abscissa : rain.
perfusion pressure and b l o o d pressure to noradrenaline (1 pg i.c.v.) in c o n t r o l rats and in rats p r e t r e a t e d with p h e n t o l a m i n e (100 pg i.c.v.). Intra-arterial b l o o d pressure prior to i.c.v. n o r a d r e n a l i n e a d m i n i s t r a t i o n in c o n t r o l rats was 109 + 10 m m Hg. Perfusion pressure was 157 -+ 15 m m Hg and h e a r t rate was 326 + 14 beats/min. Following i.c.v, n o r a d r e n a l i n e in c o n t r o l rats, b l o o d pressure fell significantly (p 0.05) f r o m pre-injection levels b y 7 + 3 m m Hg at 5 min, b y 12 + 4 m m Hg at 10 min and b y 13 -+ 3 m m Hg at 20 min. In 3 rats an initial rise in b l o o d pressure ( b e t w e e n 5 and 10 m m Hg) o f 1 - 3 min d u r a t i o n was observed. A f t e r 30 min, b l o o d pressure had n o t r e t u r n e d to pre-injection levels in 3 animals. H e a r t rate did n o t change significantly f r o m the pre-injection baseline in c o n t r o l rats, a fall o f 10 + 6 b e a t s / m i n being r e c o r d e d at 5 min and o f 1 + 2 b e a t s / m i n at 10 min. T h e significant (p < 0.01) falls in perfusion pressure in response to i.c.v, n o r a d r e n a l i n e in c o n t r o l rats always began b e t w e e n 45 and 90 sec a f t e r drug a d m i n i s t r a t i o n and r e t u r n e d to pre-injection levels w i t h i n 20 min. At 2 min, p e r f u s i o n pressure had fallen b y 10 + 1 m m Hg, at 5 m i n b y 8 - + 2 m m H g , atl0minby8 -+2mmHgandat15minby6+ 3mmHg. Prior t o i.c.v, n o r a d r e n a l i n e a d m i n i s t r a t i o n in rats p r e t r e a t e d with i.c.v, p h e n t o l a m i n e , b l o o d pressure was 87 + 10 m m Hg, p e r f u s i o n pressure was 111 + 10 m m Hg and h e a r t rate was 370 + 14 b e a t s / m i n . N o r a d r e n a l i n e (1 pg i.c.v.) i n d u c e d n o significant change in b l o o d pressure, p e r f u s i o n pressure and h e a r t rate in these rats. Perfusion pressure fell b y 6 m m Hg at 2 min and 10 m m Hg at 5 min in response to i.c.v, n o r a d r e n a l i n e in t h e atropinised hind limb p r e p a r a t i o n . T h e dose o f a t r o p i n e used b l o c k e d the d i r e c t vasodilation i n d u c e d in the hind limb b y a c e t y l c h o l i n e (0.1 pg i.a.).
3.4. Responses to i.c.v, phentolamine Intra-arterial b l o o d pressure in 10 rats p r i o r t o i.c.v, p h e n t o l a m i n e was 91 + 7 m m Hg. Per-
HINDLIMB VASCULAR BED RESPONSES TO ICV DRUG ADMINISTRATION
100 ~ ~ ==
80 L
~
............... ,,,,,~-,-,-r
,
,
I
~
TnlTrrrrmT~1~T,.i,i,~,,~
~ _ _ J
L
I
tcv NA(1,~g)
-!
,
,
J
,
I
,
I
icvphenlolomine(lOO#,g)
icy soline (lOp I)
'
17
tcv NA(1)J.g)
,
,
,
1
1
O
...o...----
300L I
I
I
I
I
T
I
I
I
I
I
I
I
I
I
I
I
I
I
I
i
i
i
i
i
i
i
I
i
i
i
i
i
l
I
i
minutes
Fig. 2. P e r f u s i o n pressure, arterial b l o o d pressure a n d h e a r t rate r e s p o n s e s of an a n i m a l to 1 pg i.c.v, n o r a d r e n a l i n e (NA) b e f o r e a n d a f t e r 100 pg i.c.v, p h e n t o l a m i n e , a n d to 10 pl i.c.v, saline. H e a r t rate readings were t a k e n at t h e p o i n t s i n d i c a t e d . A r r o w s i n d i c a t e t i m e at w h i c h drugs were a d m i n i s t e r e d .
fusion pressure was 139 + 12 mm Hg and heart rate averaged 348 + 14 beats/min. The responses of perfusion pressure, blood pressure and heart rate to i.c.v, injection of phentolamine in 1 preparation is presented in fig. 2 and the results of 10 experiments summarised in fig. 4. Following phentolamine (100 #g i.c.v.) blood pressure fell significantly (p < 0.001) by 22 + 3 m m H g a t 2 m i n , b y 2 4 + 3 m m H g at 5 min, by 26 + 4 mm Hg at 10 min and at 20 min was still depressed by 22 + 4 mm Hg (see fig. 4). Heart rate rose gradually after i.c.v, phentolamine injection. At 5 min it had increased by 15 + 10 beats/min at 10 min by 18 + 8 beats/min and at 15 min by 26 + 12 beats/ min above the pre-injection rate. Phentolamine (100 #g i.c.v.) induced a
biphasic response of perfusion pressure. There was an initial rise of 7--8 min duration (the time taken for blood from the animal to reach the isolated hind limb). This was followed by a fall in perfusion pressure to below pre-injection levels. At 2 min perfusion pressure had risen significantly (p < 0.01) by 6 + 1 mm Hg and at 5 min by 9 + 2 mm Hg (p < 0.01), but after 10 min it had fallen 8 + 3 mm Hg below the pre-injection level. At 20 min perfusion pressure was still reduced by 9 + 2 mm Hg. Prior to i.c.v, phentolamine, i.a. noradrenaline (0.1 pg) into the hind limb circulation increased the hind limb perfusion pressure by 43 + 4 mm Hg; this constrictor response was significantly reduced (p < 0.01} to 28 + 3 mm Hg when i.a. noradrenaline was injected between 12 and 25 min after i.c.v, phentolamine.
18
J.M. E L L I O T T , D.W.J. C L A R K
I0
5I 0
1
~ss~s~ ~
-5 -10
0 -5 -10 -15
-15
10 5
0 -5 40 -15
0
-20
-5
-25
-I0
-30
-15 -2[
3O 2O 1
Fig. 3. Changes in perfusion pressure ( m m Hg, upper ordinate) and arterial blood pressure (lower ordinate, m m Hg) in response to 1 pg i.c.v, noradrenaline (NA, at arrows): 8 control rats (mean weight of these rats 285 -+ 5 g, []); 5 rats (mean weight of these rats was 295 +- 7 g) pretreated with 100 pg i.c.v, p e n t o l a m i n e (A). Abscissa: min. Each s y m b o l represents the mean. Vertical bars s h o w the S.E.M. Responses of perfusion pressure differ significantly (p < 0.05) b e t w e e n the groups of animals f r o m 1--10 min after drug administration. Arterial blood pressure responses are significantly different after 7 min.
3.5. Responses to i.c.v, d, l-propranolol Intra-arterial blood pressure in 7 rats prior to i.c.v, d,l-propranolol was 125 + 5 mm Hg. Perfusion pressure was 145 + 3 mm Hg and heart rate averaged 296 + 14 beats/min. Fig. 5 shows the mean responses of perfusion pressure, blood pressure and heart rate t o i.c.v. injection o f propranolol. Following propranolol (100 pg i.c.v.) blood pressure fell significantly (p < 0.05) by 8 + 3 mm Hg at 2 min, by 24 + 6 m m Hg at 5 min, by 40 + 6 mm Hg at 10 min and at 20 min was still depressed by 42 + 8 m m Hg (fig. 5). Heart rate fell significantly (p < 0.001)
. . . . . . . 5. .
lo ....
1'5 ....
z'o
Fig. 4. Changes in perfusion pressure (ram Hg, upper ordinate), arterial b l o o d pressure ( m m Hg, middle ordinate) and heart rate (beats/min) in response to 100 pg i.c.v, p h e n t o l a m i n e (at arrows) in 10 rats. Abscissa: rain. Each s y m b o l represents the mean. Vertical bars show the S.E.M. Mean weight o f these 10 rats was 297 _+ 6 g.
after i.c.v, propranolol injection. At 5 min it had decreased by 43 + 6 beats/min, at 10 min by 55 + 7 beats/min and at 20 min by 71 + 7 beats/min. Following propranolol injection (100 pg i.c.v.) perfusion pressure in the vascularly isolated hind limb did n o t change significantly for 10 min b u t t hen fell significantly (p < 0.001). At 14 min perfusion pressure had fallen by 7 -+ 1 mm Hg and at 20 min by 10 + 1 m m Hg. At the end of these experiments section o f the femoral and sciatic nerves to the isolated hind limb was followed by an immediate fall in perfusion pressure of 55 + 3 m m Hg.
H I N D L I M B V A S C U L A R BED R E S P O N S E S TO ICV D R U G A D M I N I S T R A T I O N
-Z5 -30 -35 -40
o~ -I0 -ZO !
-30 -40 F -50 -60 -70
-80 i
0
i
J
i
h
i
i
5
i
i
t
~
10
,
r
i
i
i
15
i
i
i
i
p
20
Fig. 5. Changes in perfusion pressure ( m m Hg, upper ordinate), arterial blood pressure ( m m Hg, middle ordinate and heart rate (beats/min, lower ordinate) in response to 100 pg i.c.v, d,l-propranoiol (at arrows) in 7 rats. Each s y m b o l represents the mean. Vertical bars show the S.E.M. Mean weight of these 7 rats was 259 + 6 g.
4. Discussion Examination of the brain after i.c.v, administration of iophendylate or of indian ink confirmed that the right lateral ventricle of all rats used in perfusion experiments had been accurately cannulated. Furthermore, the fall in perfusion pressure that accompanied the pressor response to i.v. noradrenaline indicated that nervous pathways were functional. This fall in perfusion pressure and also the reflex bradycardia accompanying the blood pressure rise provided evidence that reflex
19
pathways were well preserved under the anaesthetic used. Falls in systemic blood pressure and heart rate have been demonstrated following i.c.v. noradrenaline in the anaesthetised dog (McCubbin et al., 1960; Bhargava et al., 1972); the anaesthetised cat (Nashold et al., 1962; Share and Melville, 1963; Smookler et al., 1966) and conscious cat (Day and Roach, 1974); the anaesthetised rat (Baum and Shropshire, 1973; Struyker Boudier et al., 1974) and the anaesthetised rabbit (Chalmers and Reid, 1972). Though the magnitude of the falls in intra-arterial blood pressure and heart rate obtained following i.c.v, noradrenaline injection are small, the responses observed confirm the findings of the above workers. Indeed, the fall in blood pressure observed following i.c.v, noradrenaline is slightly larger than that observed by Baum and Shropshire (1973) who injected the same dose into rats anaesthetised with Dial--urethane solution. The latency of these falls is also similar to those reported. The significant increase in heart rate observed at 10 and 15 min after i.c.v, phentolamine (see fig. 4) is at variance with the literature. Finch et al. (1975) reported t h a t i.c.v. phentolamine (200 pg) lowered intra-arterial blood pressure and heart rate in conscious DOCA/saline hypertensive rats. Intracisternal administration of this same dose was found to reduce blood pressure in rats anaesthetised with urethane (Bogaievsky et al., 1974). In vagotomised rats anaesthetised with chloralose and urethane, intracisternally administered phentolamine induced dose related decreases in blood pressure and heart rate (Ito and Schanberg, 1974). Bogaievsky et al. (1974) also reported that intracisternal phentolamine lowered blood pressure in rabbits anaesthetised with urethane but had no effect on blood pressure and heart rate in dogs anaesthetised with sodium pentobarbitone. The responses of perfusion pressure facilitate interpretation of the changes in blood pressure and heart rate observed following i.c.v, noradrenaline and i.c.v, phentolamine.
20 Blood was p u m p e d into the hind limb at a constant flow rate. Changes in perfusion pressure u n d er these constant flow conditions are a direct indication of any net vasodilation or vasoconstriction t ha t is occurring in the vascular bed. Because of the 7 min delay in the time taken for blood to reach the perfused limb, any changes in perfusion pressure occurring less than 7 min after i.c.v, drug administration must be mediated by the intact nerve supply to the hind limb. Following i.c.v, noradrenaline, perfusion pressure fell within 90 sec. This response, also observed in an atropinised hind limb, indicates th at i.c.v, injection o f noradrenaline reduces sympathetic discharge to the hind limb with a resulting ne t vasodilatation. The magnitude o f this vasodilatation is n o t great. In one series of experiments the total contribution of nervous discharge to hind limb vascular resistance was det er m i ned by section o f nerves supplying the limb. The response to i.c.v, noradrenaline was a p p r o x i m a t e l y one sixth of the vasodilatation achieved by section o f the nerves and was n o t as great as t hat induced reflexly by pressor responses to i.v. noradrenaline. The effect of doses of noradrenaline greater than 1 pg was not investigated. After i.c.v, noradrenaline the time course o f changes in perfusion pressure and blood pressure were n o t always similar; blood pressure fell more gradually than perfusion pressure and usually remained at lowered levels after perfusion pressure had returned to the pre-injection baseline. Vasodilatation in o t her vascular beds comparable to the left hind limb must have c o n t r i b u t e d to the falls in blood pressure observed and it seems t ha t either reductions in s y m p a t h e t i c o u t f l o w to these vascular beds are m or e prolonged or t hat oth er factors besides alterations in vascular t o n e are involved in the blood pressure response. It may be t ha t cardiac stroke volume has gradually fallen, giving rise to a decrease in cardiac o u t p u t despite the unchanged heart rate. Various antihypertensive agents are
J.M. ELLIOTT, D.W.J. CLARK believed to exert at least part of their action through a central reduction in sympathetic discharge. It is thus of interest to compare the vasodilatation induced by i.c.v, noradrenaline with t hat achieved by administration of such drugs. It has been suggested that the antihypertensive action of indoramin HC1 (Wyeth) is in part centrally mediated (Baum and Shropshire, 1975). In support of this we have d e m o n s t r a t e d using the vascularly isolated rat hind limb preparation, that i.c.v. (and i.v.) administration of indoramin results in a fall of perfusion pressure part of which is nervously mediated (Elliott, 1976). The magnitude of this nervously mediated fall in perfusion pressure is similar to that resulting from i.c.v, administration of noradrenaline. It has also been suggested that part of the antihypertensive action of the fi-adrenergic recept or antagonist propranolol is mediated through central mechanisms (Garvey and Ram, 1975). In our experiments, however, we were unable to dem onst rat e a nervously mediated fall in hind limb vascular resistance after i.c.v, administration of propranolol. It is tempting to speculate that propranolol immediately leaks from the CNS after i.c.v, administration leading to the fall in heart rate being due entirely to cardiac fi-adrenoreceptor blockade and that this is responsible for the observed fall in blood pressure. However, propranolol administered i.v. at the same dose as that given i.c.v, results in a fall in blood pressure only half of that seen after i.c.v, propranolol (Clark and Elliott, unpublished observation). F u r t h e r m o r e , it is of interest that following i.c.v, propranolol there was no significant reflex increase of perfusion pressure in the vascularly isolated hind limb accompanying the fall in arterial blood pressure. The mechanism o f the hypotensive response to i.c.v, propranolol awaits clarification. The vasoconstrictor effectiveness of i.a. noradrenaline was reduced 15 min after i.c.v. phentolamine. This indicates that the secondary fall in perfusion pressure beginning 8--9 min after i.c.v, phentolamine (figs. 2 and 4) is
HINDLIMB VASCULAR BED RESPONSES TO ICV DRUG ADMINISTRATION
likely to be due to leakage of the centrally administered drug into the systemic circulation and thence through the perfusion circuit to reach the isolated hind limb at least 7 min later. After such leakage from the CNS phentolamine would rapidly reach vascular beds other than the isolated hind limb and it is probably t h a t vasodilatation in these beds contributes to the observed fall in blood pressure. The tachycardia observed following i.c.v. phentolamine may be due to direct actions of phentolamine circulating in the blood. Leakage of drugs from the cerebral ventricular system into which they are administered is an inherent methodological problem of investigations into central mechanisms of cardiovascular control in that the extent of this leakage is an u n k n o w n variable. Prior i.c.v, injection of phentolamine appeared to block the nervously mediated vasodilatation induced by i.c.v, noradrenaline and it is possible that this inhibition arises from an interaction of the two drugs on central a-adrenergic receptors. However, leakage of phentolamine into the peripheral circulation and the difference in perfusion pressure levels in the phentolaminetreated and control groups do not enable a clear interpretation of this finding. In contrast to the lack of a nervously mediated response of the vascularly isolated hind limb after i.c.v, propranolol there was a nervously mediated response of the hind limb vascular bed following i.c.v, injection of both noradrenaline and phentolamine. Whereas there was an initial vasoconstriction after i.c.v, phentolamine i.c.v, noradrenaline was followed by a vasodilatation in the vascularly isolated hind limb. It is not known whether the vasoconstriction is mediated through an action of phentolamine on central adrenergic receptors or is a reflex response to the fall in systemic blood pressure by i.c.v, phentolamine. However, the technique described in this paper has indicated that i.c.v, noradrenaline and i.c.v, phentolamine lower blood pressure through different mechanisms: the action of noradrenaline is within the central nervous system but that of phentolamine is likely to
21
be peripheral. The preparation described in this paper will thus be of use in clarifying the sites of action of drugs t h o u g h t to lower blood pressure wholly or partly through central mechanisms.
Acknowledgement This work was supported by the Medical Research Council of New Zealand.
References Baum, T. and A.T. Shropshire, 1970, Inhibition of spontaneous sympathetic nerve activity by the antihypertensive agent Wy-8678, Neuropharmacology 9, 503. Baum, T. and A.T. Shropshire, 1973, Reduction of sympathetic outflow by central administration of L-dopa, dopamine and norepinephrine, Neuropharmacology 12, 49. Baum, T. and A.T. Shropshire, 1975, Central and peripheral contribution to the antihypertensive action of indoramin, European J. Pharmacol. 32, 30. Bhargava, K.P., N. Mishra and K.K. Tangri, 1972, An analysis of central adrenoreceptors for control of cardiovascular function, Brit. J. Pharmacol. 45, 596. Bogaievsky, D., Y. Bogaievsky, D. Tsoucaris-Kupfer and H. Schmitt, 1974, Blockade of the central hypotensive effect of clonidine by a-adrenoreceptor antagonists in rats, rabbits and dogs, Clin. Exptl. Pharmacol. Physiol. 1, 527. Chalmers, J.P., 1975, Brain amines and models of experimental hypertension, Circulation Res. 36, 469. Chalmers, J.P. and J.L. Reid, 1972, Participation of central noradrenergic neurons in arterial baroreceptor reflexes in the rabbit. A study with intracisternally administered 6°hydroxydopamine, Circulation Res. 31, 789. Day, M.D. and A.G. Roach, 1974, Central adrenoreceptors and the control of arterial blood pressure, Clin. Exptl. Pharmacol. Physiol. 1,347. Elliott, J.M., 1976, A centrally mediated vasodilator effect of indoramin, Proc. Univ. Otago Med. Sch. 54, 67. Elliott, J.M. and D.W.J. Clark, 1976, Vasodilatation induced in the blood-perfused hind limb by central administration of noradrenaline, Proc. Univ. Otago Med. Sch. 54, 38.
22 Finch, L., R.E. Buckingham, R. Moore and T.J. Bucher, 1975, Evidence for a central ~-sympathomimetic action of clonidine in the rat, J. Pharm. Pharmacol. 27, 181. Garvey, H.L. and N. Ram, 1975, Centrally induced hypotensive effects of ~-adrenergic blocking drugs, European J. Pharmacol. 33, 283. Haeusler, G., 1975, Cardiovascular regulation by central adrenergic mechanisms and its alteration by hypotensive drugs, Circulation Res, 36, Suppl. 1, 223. Hayden, J.F., L.R. Johnson and R.P. Maickel, 1966, Construction and implantation of a permanent cannula for making injections into the lateral ventricle of the rat brain, Life Sci. 5, 1509. Ito, A. and S.M. Schanberg, 1974, Maintenance of tonic vasomotor activity by alpha and beta adrenergic mechanisms in medullary cardiovascular centers, J. Pharmacol. Exptl. Therap. 189,392. Laverty, R., 1962, Relation in rat hind-limb blood vessels between nervous vasomotor tone and the response to vasoconstrictor drugs, Brit. J. Pharmacol. 18,451. Laverty, R., 1973, The mechanisms of action of some antihypertensive drugs, Brit. Med. Bull. 29, 152. McCubbin, J.W., Y. Kaneko and I.H. Page, 1960, Ability of serotonin and norepinephrine to mimic the central effects of reserpine on vasomotor activity, Circulation Res. 8, 849. Nashold, B.S., E. Mannarino and M. Wunderlich,
J.M. ELLIOTT, D.W.J. CLARK 1962, Pressor-depressor blood pressure responses in the cat after intraventricular injection of drugs, Nature, London 193, 1297. Pichler, L. and W. Kobinger, 1976, Decrease of preganglionic sympathetic discharges by intracisternally administered ~-adrenergic imidazoline compounds, Naunyn-Schmiedeb. Arch. Pharmacol. 293, R16. Schmitt, H., and H. Schmitt, 1969, Localisation of the hypertensive effect of 2o(2-6-dichlorophenylamino)-2-imidazoline hydrochloride (St 155, Catapresan), European J. Pharmacol. 6, 8. Scriabine, A., B.V. Clineschmidt and C.S. Sweet, 1976, Central noradrenergic control of blood pressure, Ann. Rev. Pharmacol. Toxicol. 16, 113. Share, N.N: and K.I. Melville, 1963, Centrally mediated sympathetic cardiovascular responses induced by intraventricular norepinephrine, J. Pharmacol. Exptl. Therap. 141, 15. Smookler, H.H., W.B. Severs, W.J. Kinnard and J.P. Buckley, 1966, Centrally mediated cardiovascular effects of angiotensin II, J. Pharmacol. Exptl. Therap. 153, 485. Struyker Boudier, H.A.J., G. Smeets, G. Brouwer and J.M. Van Rossum, 1974, Central and peripheral alpha adrenergic activity of imidazoline derivatives, Life Sci. 15,887. Van Zwieten, P.A., 1973, The central action of antihypertensive drugs mediated via central ~-receptors, J. Pharm. Pharmacol. 25, 89.
13
C H A N G E S IN R A T H I N D L I M B V A S C U L A R R E S I S T A N C E F O L L O W I N G INTRACEREBROVENTRICULAR DRUG ADMINISTRATION JOHN M. ELLIOTT and DAVID W.J. CLARK Department of Pharmacology, Otago Medical School, Dunedin, New Zealand
Received 8 February 1977, revised MS received 13 April 1977, accepted 17 May 1977
J.M. ELLIOTT and D.W.J. CLARK, Changes in rat hind limb vascular resistance following intracerebroventricular drug administration, European J. Pharmacol. 45 (1977) 13--22. The right hind limbs of rats (which had previously implanted intraventricular guide cannulae) were isolated from the systemic circulation, but with the nerves to the limb remaining intact, and perfused using a constant output blood pump. Using this preparation, changes in vascular resistance, blood pressure and heart rate were monitored following injection of noradrenaline, phentolamine and propranolol into the lateral cerebral ventricles (i.c.v.) of rats anaesthetised with a-chloralose. All three drugs lowered blood pressure. Noradrenaline administered i.c.v, induced a nervously mediated vasodilatation and an insignificant fall in heart rate whereas i.c.v, phentolamine administration was followed by a nervously mediated vasoconstriction in the isolated hind limb and a gradual rise in heart rate. After i.c.v, administration of propranolol there was no evidence of an immediate nervously mediated vasodilatation but heart rate fell significantly. Following i.c.v, phentolamine or propranolol vasodilatation did not occur in the hind limb until after the time taken for circulating blood ro reach the isolated vascular bed. The vasodilatatory and hypotensive responses to i.c.v, noradrenaline were not evident following prior i.c.v, injection of phentolamine. These results indicate the suitability of this preparation for investigations of central actions of other drugs. Antihypertensive agents Perfusion pressure
I.c.v. injections Heart rate
Blood pressure Noradrenaline
1. I n t r o d u c t i o n Several drugs have r e c e n t l y b e e n introduced into antihypertensive therapy which are t h o u g h t to e x e r t h y p o t e n s i v e actions b y w a y o f t h e central n e r v o u s s y s t e m . Such drugs i n c l u d e reserpine, a - m e t h y l d o p a , c l o n i d i n e , i n d o r a m i n and f l - a d r e n o r e c e p t o r b l o c k i n g drugs (Laverty, 1 9 7 3 ; van Zwieten, 1 9 7 3 ; D a y and R o c h , 1 9 7 4 ; B a u m and S h r o p s h i r e , 1 9 7 5 ; Scriabine et al., 1 9 7 6 ) . The investigation o f these drugs has p r o m p t e d intensive research i n t o the n e u r o p h y s i o l o g i c a l basis o f the central c o n t r o l o f cardiovascular f u n c t i o n i n g . The greatest b o d y o f evidence so far p r e s e n t e d is t h a t i m p l i c a t i n g e n d o g e n o u s n o r a d r e n a l i n e in the central c o n t r o l o f arterial b l o o d pressure
Phentolamine
Propranolol
(Day and R o a c h , 1974; Chalmers, 1 9 7 5 ; Haeusler, 1975). In m o s t earlier investigations, b l o o d pressure and h e a r t rate have b e e n m o n i t o r e d following the injection o f n o r a d r e n a l i n e into the cerebral v e n t r i c u l a r s y s t e m . Generally such a d m i n i s t r a t i o n has i n d u c e d a fall in b o t h b l o o d pressure and h e a r t rate. In r e c e n t years s o m e researchers have m o n i t o r e d electrical activity in a s e c t i o n e d s p l a n c h n i c nerve ( S c h m i t t and S c h m i t t , 1 9 6 9 ; B a u m and Shropshire, 1 9 7 0 , ] 9 7 3 ; Pichler and Kobinger, 1 9 7 6 ) . In this w a y t h e y have estim a t e d general changes in s y m p a t h e t i c o u t f l o w f r o m the central n e r v o u s s y s t e m . These studies have s h o w n t h a t c e n t r a l l y a d m i n i s t e r e d n o r a d r e n a l i n e decreases i m p u l s e flow along
14
the sectioned sympathetic nerve trunk. However, such measurement of electrical activity is not specific for impulse flow which is destined to supply the walls of blood vessels. Use of the innervated but vascularly isolated rat hind limb preparation {Laverty, 1962), perfused with blood at a constant rate, enables observation of vascular end organ responses to alteration in autonomic nervous activity induced by central administration of drugs. Furthermore, the incorporation of a delay coil into the perfusion circuit enables clear separation of nervously mediated and humorally mediated changes in vascular resistance. This paper describes response of perfusion pressure (proportional to the vascular resistance of the hind limb vascular bed), blood pressure and heart rate to centrally administered noradrenaline, phentolamine and propranolol. A preliminary report of some of the work has been given to the Otago Medical School Research Society (Elliott and Clark, 1976). 2. Materials and methods
J.M. E L L I O T T , D.W.J. C L A R K
2.2. The isolated hind limb preparation The left femoral vein was cannulated under ether anaesthesia, anaesthesia was maintained with a-chloralose {80 mg/kg i.v.) and the animal's trachea cannulated. The right hind limb was isolated from the main circulation (Laverty, 1962) but the femoral and sciatic nerves and the femoral vein were left intact. The preparation was perfused with blood, at the rate of 1 ml/min, by means of a non-haemolysing, constant o u t p u t blood pump constructed in the workshops of the Wellcome Medical Research Institute, Dunedin, N.Z. This pump and the tubing making up the perfusion circuit were primed with blood from a donor rat. A delay coil in the perfusion circuit ensured that blood reached the isolated hind limb between 6 and 7 min after being drawn from the opposite femoral artery. Blood pressure and perfusion pressure (proportional to vascular resistance) were monitored using pressure transducers and a pen recorder. Heart rates were determined from the ECG.
2.1. Implantation o f i.c.v, guide cannulae
2.3. Experimental procecure
Male Wistar rats (250--350 g) were used. Each rat was anaesthetised with sodium pentobarbitone (20 mg/kg i.p.) and ketamine (100 mg/kg i.p. injected contralaterally 15 min later) and placed in a stereotaxic apparatus. The skull surface was exposed and cleared, and a hole was drilled through the left parietal bone to position an anchoring screw. A second hole was then drilled 1 mm lateral and caudal to the bregma and a 5 mm guide cannula, similar to that described by Hayden et al. {1966) was lowered through this hole so that its bevelled tip came to lie within the right lateral ventricle. The perspex section of the cannula was then cemented to the anchoring screw and to the skull surface. The wound was sutured and the rat allowed to recover for at least one week before being used for perfusion experiments.
2.3.1. Test for nerve function As a test for the integrity of the autonomic nerve supply to the hind limb, i.v. noradrenaline (0.5 pg/kg) was injected at the beginning of every experiment and responses of blood pressure, perfusion pressure and heart rate were observed. 2.3.2. I.c.v. injections A length of 27 gauge needle was inserted into a short length of polyethylene tubing, the other end of which was attached to a 50 pl Hamilton syringe. The syringe, tubing and needle were then filled with the solution to be injected. The 27 gauge needle was then inserted into the chronically implanted guide cannula to a depth of 1 cm so that its tip was at the tip of the metal section of the guide cannula. In this way drugs could be injected
HINDLIMB V A S C U L A R BED RESPONSES TO ICV DRUG ADMINISTRATION
directly into the cerebral ventricle. Preliminary experiments were performed to find the minimum dose of noradrenaline which when administered i.c.v, produced an effect on hind limb perfusion pressure. After a stable perfusion pressure had been recorded for at least 10 min, 10 #1 of either noradrenaline (1 #g in 0.9% NaC1) or 0.9% NaC1 was injected i.c.v, at intervals of 30 min. Responses of perfusion pressure, blood pressure and heart rate to 2 i.c.v, injections of noradrenaline were recorded in 5 rats. In another 3 rats responses to 1 i.c.v, injection of noradrenaline were recorded. These 8 rats are referred to as "control rats" in the Results section. In 3 of the above preparations, and in a further 7 rats which had received no other centrally administered drug, phentolamine (100 pg) was injected i.c.v. Responses of perfusion pressure, blood pressure and heart rate were recorded. When perfusion pressure had again stabilized (10--15 min after the injection of phentolamine), 0.9% N a C 1 (10 pl) was injected to ensure no phentolamine remained in the guide cannula. At least 10 min later noradrenaline (1 pg in 10 pl 0.9% NaC1) was injected i.c.v, in the first group of 3 rats and in 2 further rats from the second group, and the responses o f perfusion pressure, blood pressure and heart rate were monitored. In a further 7 rats which had received no other centrally administered drug, propranolol (100 pg in 10 pl 0.9% NaC1) was injected i.c.v. Responses of perfusion pressure, blood pressure and heart rate were recorded. At least 60 min after i.c.v, injection of propranolol the femoral and sciatic nerves supplying the vascularly isolated hind limbs were sectioned and the resulting fall in perfusion pressure recorded.
2.3.3. Effects o f i.a. noradrenaline The influence of i.c.v, phentolamine on the pressor effectiveness of i.a. noradrenaline in the isolated hind limb was investigated in 6 preparations. Prior to i.c.v, injection of phentolamine, 0.1 pg noradrenaline (20 pl of 5
15
pg/ml in 0.9% NaC1 solution) was injected i.a. 2--3 times and perfusion pressure responses were averaged. At least 12 min after phentolamine (100 pg i.c.v.) two further doses of noradrenaline (0.1 pg i.a.) were given. Responses were again averaged and the pre- and post-phentolamine responses compared by the paired t-test.
2.3.4. Atropinised hind limb In 1 further experiment acetylcholine (0.1 pg in 100 pl) was injected into the hind limb (i.a.). After 10 min, atropine (1 mg in 100 pl) was injected i.a., followed by acetylcholine (0.1 pg i.a.). 5 min later, noradrenaline (1 #g i.c.v.) was administered and responses were recorded for 7 min, at which time a further dose of acetylcholine (0.1 pg i.a.) was injected. 2.3.5. I.c.v. indian ink At the completion of every experiment, indian ink (10 pl) was injected i.c.v. The brain was immediately removed from the skull and sectioned for examination of the distribution of the ink. 2. 4. Drugs The following drugs were administered: atropine sulphate, MacFarlan Smith Ltd.; noradrenaline bitartrate (Levophed), Winthrop; phentolamine mesylate (Rogitine), Ciba; d,l-propranolol hydrochloride, Sigma.
3. Results
3.1. Results o f distribution o f iophendylate and indian ink X-ray examination confirmed that the right lateral ventricle had been accurately cannulated. The radio-opaque iophendylate outlined the semicircular shaped lateral ventricles and the third ventricle below. The bony structures of the ear masked the hind brain regions and so the disposition of the iophendylate
16
J.M. ELLIOTT, D.W.J. CLARK
c o u l d n o t be f u r t h e r d e t e r m i n e d . Sectioning o f the brain to e x a m i n e the dist r i b u t i o n o f i.c.v, indian ink always s h o w e d the d y e t o be present in b o t h lateral ventricles and as far caudal as the f o u r t h ventricle and cisterna magna. Usually some indian ink had diffused into the s u b a r a c h n o i d space a r o u n d the spinal c o r d at the level o f C2 and C3.
3.2. Response to i.v. noradrenaline I.v. n o r a d r e n a l i n e (0.5 pg/kg), injected at the beginning o f e v e r y e x p e r i m e n t , i n d u c e d a rise in b l o o d pressure averaging 50 m m Hg. This pressor r e s p o n s e was a c c o m p a n i e d b y a (reflex) fall in p e r f u s i o n pressure o f 1 5 - - 3 0 m m Hg in the vascularly isolated i n n e r v a t e d hind limb and a fall in h e a r t rate (fig. 1).
3.3. Responses to i.c.v, noradrenaline Sections o f tracing o b t a i n e d d u r i n g an e x p e r i m e n t are p r e s e n t e d in fig. 2. Fig. 3 shows a c o m p a r i s o n o f the m e a n responses o f
'8°F 160F 140 F
120F I00L-
160F
2E I
I
I
I
I
I
I
I
I
Fig. 1. Arterial blood pressure (upper level, mm Hg), perfusion pressure (middle level, mm Hg) and heart
rate (lower level, beats/min) responses of an animal to 0.5 ~g/kg i.v. noradrenaline (NA, at arrows). The regular transient falls in perfusion pressure and increases in arterial blood pressure are a pump artefact. Heart rate was recorded at the points indicated. Abscissa : rain.
perfusion pressure and b l o o d pressure to noradrenaline (1 pg i.c.v.) in c o n t r o l rats and in rats p r e t r e a t e d with p h e n t o l a m i n e (100 pg i.c.v.). Intra-arterial b l o o d pressure prior to i.c.v. n o r a d r e n a l i n e a d m i n i s t r a t i o n in c o n t r o l rats was 109 + 10 m m Hg. Perfusion pressure was 157 -+ 15 m m Hg and h e a r t rate was 326 + 14 beats/min. Following i.c.v, n o r a d r e n a l i n e in c o n t r o l rats, b l o o d pressure fell significantly (p 0.05) f r o m pre-injection levels b y 7 + 3 m m Hg at 5 min, b y 12 + 4 m m Hg at 10 min and b y 13 -+ 3 m m Hg at 20 min. In 3 rats an initial rise in b l o o d pressure ( b e t w e e n 5 and 10 m m Hg) o f 1 - 3 min d u r a t i o n was observed. A f t e r 30 min, b l o o d pressure had n o t r e t u r n e d to pre-injection levels in 3 animals. H e a r t rate did n o t change significantly f r o m the pre-injection baseline in c o n t r o l rats, a fall o f 10 + 6 b e a t s / m i n being r e c o r d e d at 5 min and o f 1 + 2 b e a t s / m i n at 10 min. T h e significant (p < 0.01) falls in perfusion pressure in response to i.c.v, n o r a d r e n a l i n e in c o n t r o l rats always began b e t w e e n 45 and 90 sec a f t e r drug a d m i n i s t r a t i o n and r e t u r n e d to pre-injection levels w i t h i n 20 min. At 2 min, p e r f u s i o n pressure had fallen b y 10 + 1 m m Hg, at 5 m i n b y 8 - + 2 m m H g , atl0minby8 -+2mmHgandat15minby6+ 3mmHg. Prior t o i.c.v, n o r a d r e n a l i n e a d m i n i s t r a t i o n in rats p r e t r e a t e d with i.c.v, p h e n t o l a m i n e , b l o o d pressure was 87 + 10 m m Hg, p e r f u s i o n pressure was 111 + 10 m m Hg and h e a r t rate was 370 + 14 b e a t s / m i n . N o r a d r e n a l i n e (1 pg i.c.v.) i n d u c e d n o significant change in b l o o d pressure, p e r f u s i o n pressure and h e a r t rate in these rats. Perfusion pressure fell b y 6 m m Hg at 2 min and 10 m m Hg at 5 min in response to i.c.v, n o r a d r e n a l i n e in t h e atropinised hind limb p r e p a r a t i o n . T h e dose o f a t r o p i n e used b l o c k e d the d i r e c t vasodilation i n d u c e d in the hind limb b y a c e t y l c h o l i n e (0.1 pg i.a.).
3.4. Responses to i.c.v, phentolamine Intra-arterial b l o o d pressure in 10 rats p r i o r t o i.c.v, p h e n t o l a m i n e was 91 + 7 m m Hg. Per-
HINDLIMB VASCULAR BED RESPONSES TO ICV DRUG ADMINISTRATION
100 ~ ~ ==
80 L
~
............... ,,,,,~-,-,-r
,
,
I
~
TnlTrrrrmT~1~T,.i,i,~,,~
~ _ _ J
L
I
tcv NA(1,~g)
-!
,
,
J
,
I
,
I
icvphenlolomine(lOO#,g)
icy soline (lOp I)
'
17
tcv NA(1)J.g)
,
,
,
1
1
O
...o...----
300L I
I
I
I
I
T
I
I
I
I
I
I
I
I
I
I
I
I
I
I
i
i
i
i
i
i
i
I
i
i
i
i
i
l
I
i
minutes
Fig. 2. P e r f u s i o n pressure, arterial b l o o d pressure a n d h e a r t rate r e s p o n s e s of an a n i m a l to 1 pg i.c.v, n o r a d r e n a l i n e (NA) b e f o r e a n d a f t e r 100 pg i.c.v, p h e n t o l a m i n e , a n d to 10 pl i.c.v, saline. H e a r t rate readings were t a k e n at t h e p o i n t s i n d i c a t e d . A r r o w s i n d i c a t e t i m e at w h i c h drugs were a d m i n i s t e r e d .
fusion pressure was 139 + 12 mm Hg and heart rate averaged 348 + 14 beats/min. The responses of perfusion pressure, blood pressure and heart rate to i.c.v, injection of phentolamine in 1 preparation is presented in fig. 2 and the results of 10 experiments summarised in fig. 4. Following phentolamine (100 #g i.c.v.) blood pressure fell significantly (p < 0.001) by 22 + 3 m m H g a t 2 m i n , b y 2 4 + 3 m m H g at 5 min, by 26 + 4 mm Hg at 10 min and at 20 min was still depressed by 22 + 4 mm Hg (see fig. 4). Heart rate rose gradually after i.c.v, phentolamine injection. At 5 min it had increased by 15 + 10 beats/min at 10 min by 18 + 8 beats/min and at 15 min by 26 + 12 beats/ min above the pre-injection rate. Phentolamine (100 #g i.c.v.) induced a
biphasic response of perfusion pressure. There was an initial rise of 7--8 min duration (the time taken for blood from the animal to reach the isolated hind limb). This was followed by a fall in perfusion pressure to below pre-injection levels. At 2 min perfusion pressure had risen significantly (p < 0.01) by 6 + 1 mm Hg and at 5 min by 9 + 2 mm Hg (p < 0.01), but after 10 min it had fallen 8 + 3 mm Hg below the pre-injection level. At 20 min perfusion pressure was still reduced by 9 + 2 mm Hg. Prior to i.c.v, phentolamine, i.a. noradrenaline (0.1 pg) into the hind limb circulation increased the hind limb perfusion pressure by 43 + 4 mm Hg; this constrictor response was significantly reduced (p < 0.01} to 28 + 3 mm Hg when i.a. noradrenaline was injected between 12 and 25 min after i.c.v, phentolamine.
18
J.M. E L L I O T T , D.W.J. C L A R K
I0
5I 0
1
~ss~s~ ~
-5 -10
0 -5 -10 -15
-15
10 5
0 -5 40 -15
0
-20
-5
-25
-I0
-30
-15 -2[
3O 2O 1
Fig. 3. Changes in perfusion pressure ( m m Hg, upper ordinate) and arterial blood pressure (lower ordinate, m m Hg) in response to 1 pg i.c.v, noradrenaline (NA, at arrows): 8 control rats (mean weight of these rats 285 -+ 5 g, []); 5 rats (mean weight of these rats was 295 +- 7 g) pretreated with 100 pg i.c.v, p e n t o l a m i n e (A). Abscissa: min. Each s y m b o l represents the mean. Vertical bars s h o w the S.E.M. Responses of perfusion pressure differ significantly (p < 0.05) b e t w e e n the groups of animals f r o m 1--10 min after drug administration. Arterial blood pressure responses are significantly different after 7 min.
3.5. Responses to i.c.v, d, l-propranolol Intra-arterial blood pressure in 7 rats prior to i.c.v, d,l-propranolol was 125 + 5 mm Hg. Perfusion pressure was 145 + 3 mm Hg and heart rate averaged 296 + 14 beats/min. Fig. 5 shows the mean responses of perfusion pressure, blood pressure and heart rate t o i.c.v. injection o f propranolol. Following propranolol (100 pg i.c.v.) blood pressure fell significantly (p < 0.05) by 8 + 3 mm Hg at 2 min, by 24 + 6 m m Hg at 5 min, by 40 + 6 mm Hg at 10 min and at 20 min was still depressed by 42 + 8 m m Hg (fig. 5). Heart rate fell significantly (p < 0.001)
. . . . . . . 5. .
lo ....
1'5 ....
z'o
Fig. 4. Changes in perfusion pressure (ram Hg, upper ordinate), arterial b l o o d pressure ( m m Hg, middle ordinate) and heart rate (beats/min) in response to 100 pg i.c.v, p h e n t o l a m i n e (at arrows) in 10 rats. Abscissa: rain. Each s y m b o l represents the mean. Vertical bars show the S.E.M. Mean weight o f these 10 rats was 297 _+ 6 g.
after i.c.v, propranolol injection. At 5 min it had decreased by 43 + 6 beats/min, at 10 min by 55 + 7 beats/min and at 20 min by 71 + 7 beats/min. Following propranolol injection (100 pg i.c.v.) perfusion pressure in the vascularly isolated hind limb did n o t change significantly for 10 min b u t t hen fell significantly (p < 0.001). At 14 min perfusion pressure had fallen by 7 -+ 1 mm Hg and at 20 min by 10 + 1 m m Hg. At the end of these experiments section o f the femoral and sciatic nerves to the isolated hind limb was followed by an immediate fall in perfusion pressure of 55 + 3 m m Hg.
H I N D L I M B V A S C U L A R BED R E S P O N S E S TO ICV D R U G A D M I N I S T R A T I O N
-Z5 -30 -35 -40
o~ -I0 -ZO !
-30 -40 F -50 -60 -70
-80 i
0
i
J
i
h
i
i
5
i
i
t
~
10
,
r
i
i
i
15
i
i
i
i
p
20
Fig. 5. Changes in perfusion pressure ( m m Hg, upper ordinate), arterial blood pressure ( m m Hg, middle ordinate and heart rate (beats/min, lower ordinate) in response to 100 pg i.c.v, d,l-propranoiol (at arrows) in 7 rats. Each s y m b o l represents the mean. Vertical bars show the S.E.M. Mean weight of these 7 rats was 259 + 6 g.
4. Discussion Examination of the brain after i.c.v, administration of iophendylate or of indian ink confirmed that the right lateral ventricle of all rats used in perfusion experiments had been accurately cannulated. Furthermore, the fall in perfusion pressure that accompanied the pressor response to i.v. noradrenaline indicated that nervous pathways were functional. This fall in perfusion pressure and also the reflex bradycardia accompanying the blood pressure rise provided evidence that reflex
19
pathways were well preserved under the anaesthetic used. Falls in systemic blood pressure and heart rate have been demonstrated following i.c.v. noradrenaline in the anaesthetised dog (McCubbin et al., 1960; Bhargava et al., 1972); the anaesthetised cat (Nashold et al., 1962; Share and Melville, 1963; Smookler et al., 1966) and conscious cat (Day and Roach, 1974); the anaesthetised rat (Baum and Shropshire, 1973; Struyker Boudier et al., 1974) and the anaesthetised rabbit (Chalmers and Reid, 1972). Though the magnitude of the falls in intra-arterial blood pressure and heart rate obtained following i.c.v, noradrenaline injection are small, the responses observed confirm the findings of the above workers. Indeed, the fall in blood pressure observed following i.c.v, noradrenaline is slightly larger than that observed by Baum and Shropshire (1973) who injected the same dose into rats anaesthetised with Dial--urethane solution. The latency of these falls is also similar to those reported. The significant increase in heart rate observed at 10 and 15 min after i.c.v, phentolamine (see fig. 4) is at variance with the literature. Finch et al. (1975) reported t h a t i.c.v. phentolamine (200 pg) lowered intra-arterial blood pressure and heart rate in conscious DOCA/saline hypertensive rats. Intracisternal administration of this same dose was found to reduce blood pressure in rats anaesthetised with urethane (Bogaievsky et al., 1974). In vagotomised rats anaesthetised with chloralose and urethane, intracisternally administered phentolamine induced dose related decreases in blood pressure and heart rate (Ito and Schanberg, 1974). Bogaievsky et al. (1974) also reported that intracisternal phentolamine lowered blood pressure in rabbits anaesthetised with urethane but had no effect on blood pressure and heart rate in dogs anaesthetised with sodium pentobarbitone. The responses of perfusion pressure facilitate interpretation of the changes in blood pressure and heart rate observed following i.c.v, noradrenaline and i.c.v, phentolamine.
20 Blood was p u m p e d into the hind limb at a constant flow rate. Changes in perfusion pressure u n d er these constant flow conditions are a direct indication of any net vasodilation or vasoconstriction t ha t is occurring in the vascular bed. Because of the 7 min delay in the time taken for blood to reach the perfused limb, any changes in perfusion pressure occurring less than 7 min after i.c.v, drug administration must be mediated by the intact nerve supply to the hind limb. Following i.c.v, noradrenaline, perfusion pressure fell within 90 sec. This response, also observed in an atropinised hind limb, indicates th at i.c.v, injection o f noradrenaline reduces sympathetic discharge to the hind limb with a resulting ne t vasodilatation. The magnitude o f this vasodilatation is n o t great. In one series of experiments the total contribution of nervous discharge to hind limb vascular resistance was det er m i ned by section o f nerves supplying the limb. The response to i.c.v, noradrenaline was a p p r o x i m a t e l y one sixth of the vasodilatation achieved by section o f the nerves and was n o t as great as t hat induced reflexly by pressor responses to i.v. noradrenaline. The effect of doses of noradrenaline greater than 1 pg was not investigated. After i.c.v, noradrenaline the time course o f changes in perfusion pressure and blood pressure were n o t always similar; blood pressure fell more gradually than perfusion pressure and usually remained at lowered levels after perfusion pressure had returned to the pre-injection baseline. Vasodilatation in o t her vascular beds comparable to the left hind limb must have c o n t r i b u t e d to the falls in blood pressure observed and it seems t ha t either reductions in s y m p a t h e t i c o u t f l o w to these vascular beds are m or e prolonged or t hat oth er factors besides alterations in vascular t o n e are involved in the blood pressure response. It may be t ha t cardiac stroke volume has gradually fallen, giving rise to a decrease in cardiac o u t p u t despite the unchanged heart rate. Various antihypertensive agents are
J.M. ELLIOTT, D.W.J. CLARK believed to exert at least part of their action through a central reduction in sympathetic discharge. It is thus of interest to compare the vasodilatation induced by i.c.v, noradrenaline with t hat achieved by administration of such drugs. It has been suggested that the antihypertensive action of indoramin HC1 (Wyeth) is in part centrally mediated (Baum and Shropshire, 1975). In support of this we have d e m o n s t r a t e d using the vascularly isolated rat hind limb preparation, that i.c.v. (and i.v.) administration of indoramin results in a fall of perfusion pressure part of which is nervously mediated (Elliott, 1976). The magnitude of this nervously mediated fall in perfusion pressure is similar to that resulting from i.c.v, administration of noradrenaline. It has also been suggested that part of the antihypertensive action of the fi-adrenergic recept or antagonist propranolol is mediated through central mechanisms (Garvey and Ram, 1975). In our experiments, however, we were unable to dem onst rat e a nervously mediated fall in hind limb vascular resistance after i.c.v, administration of propranolol. It is tempting to speculate that propranolol immediately leaks from the CNS after i.c.v, administration leading to the fall in heart rate being due entirely to cardiac fi-adrenoreceptor blockade and that this is responsible for the observed fall in blood pressure. However, propranolol administered i.v. at the same dose as that given i.c.v, results in a fall in blood pressure only half of that seen after i.c.v, propranolol (Clark and Elliott, unpublished observation). F u r t h e r m o r e , it is of interest that following i.c.v, propranolol there was no significant reflex increase of perfusion pressure in the vascularly isolated hind limb accompanying the fall in arterial blood pressure. The mechanism o f the hypotensive response to i.c.v, propranolol awaits clarification. The vasoconstrictor effectiveness of i.a. noradrenaline was reduced 15 min after i.c.v. phentolamine. This indicates that the secondary fall in perfusion pressure beginning 8--9 min after i.c.v, phentolamine (figs. 2 and 4) is
HINDLIMB VASCULAR BED RESPONSES TO ICV DRUG ADMINISTRATION
likely to be due to leakage of the centrally administered drug into the systemic circulation and thence through the perfusion circuit to reach the isolated hind limb at least 7 min later. After such leakage from the CNS phentolamine would rapidly reach vascular beds other than the isolated hind limb and it is probably t h a t vasodilatation in these beds contributes to the observed fall in blood pressure. The tachycardia observed following i.c.v. phentolamine may be due to direct actions of phentolamine circulating in the blood. Leakage of drugs from the cerebral ventricular system into which they are administered is an inherent methodological problem of investigations into central mechanisms of cardiovascular control in that the extent of this leakage is an u n k n o w n variable. Prior i.c.v, injection of phentolamine appeared to block the nervously mediated vasodilatation induced by i.c.v, noradrenaline and it is possible that this inhibition arises from an interaction of the two drugs on central a-adrenergic receptors. However, leakage of phentolamine into the peripheral circulation and the difference in perfusion pressure levels in the phentolaminetreated and control groups do not enable a clear interpretation of this finding. In contrast to the lack of a nervously mediated response of the vascularly isolated hind limb after i.c.v, propranolol there was a nervously mediated response of the hind limb vascular bed following i.c.v, injection of both noradrenaline and phentolamine. Whereas there was an initial vasoconstriction after i.c.v, phentolamine i.c.v, noradrenaline was followed by a vasodilatation in the vascularly isolated hind limb. It is not known whether the vasoconstriction is mediated through an action of phentolamine on central adrenergic receptors or is a reflex response to the fall in systemic blood pressure by i.c.v, phentolamine. However, the technique described in this paper has indicated that i.c.v, noradrenaline and i.c.v, phentolamine lower blood pressure through different mechanisms: the action of noradrenaline is within the central nervous system but that of phentolamine is likely to
21
be peripheral. The preparation described in this paper will thus be of use in clarifying the sites of action of drugs t h o u g h t to lower blood pressure wholly or partly through central mechanisms.
Acknowledgement This work was supported by the Medical Research Council of New Zealand.
References Baum, T. and A.T. Shropshire, 1970, Inhibition of spontaneous sympathetic nerve activity by the antihypertensive agent Wy-8678, Neuropharmacology 9, 503. Baum, T. and A.T. Shropshire, 1973, Reduction of sympathetic outflow by central administration of L-dopa, dopamine and norepinephrine, Neuropharmacology 12, 49. Baum, T. and A.T. Shropshire, 1975, Central and peripheral contribution to the antihypertensive action of indoramin, European J. Pharmacol. 32, 30. Bhargava, K.P., N. Mishra and K.K. Tangri, 1972, An analysis of central adrenoreceptors for control of cardiovascular function, Brit. J. Pharmacol. 45, 596. Bogaievsky, D., Y. Bogaievsky, D. Tsoucaris-Kupfer and H. Schmitt, 1974, Blockade of the central hypotensive effect of clonidine by a-adrenoreceptor antagonists in rats, rabbits and dogs, Clin. Exptl. Pharmacol. Physiol. 1, 527. Chalmers, J.P., 1975, Brain amines and models of experimental hypertension, Circulation Res. 36, 469. Chalmers, J.P. and J.L. Reid, 1972, Participation of central noradrenergic neurons in arterial baroreceptor reflexes in the rabbit. A study with intracisternally administered 6°hydroxydopamine, Circulation Res. 31, 789. Day, M.D. and A.G. Roach, 1974, Central adrenoreceptors and the control of arterial blood pressure, Clin. Exptl. Pharmacol. Physiol. 1,347. Elliott, J.M., 1976, A centrally mediated vasodilator effect of indoramin, Proc. Univ. Otago Med. Sch. 54, 67. Elliott, J.M. and D.W.J. Clark, 1976, Vasodilatation induced in the blood-perfused hind limb by central administration of noradrenaline, Proc. Univ. Otago Med. Sch. 54, 38.
22 Finch, L., R.E. Buckingham, R. Moore and T.J. Bucher, 1975, Evidence for a central ~-sympathomimetic action of clonidine in the rat, J. Pharm. Pharmacol. 27, 181. Garvey, H.L. and N. Ram, 1975, Centrally induced hypotensive effects of ~-adrenergic blocking drugs, European J. Pharmacol. 33, 283. Haeusler, G., 1975, Cardiovascular regulation by central adrenergic mechanisms and its alteration by hypotensive drugs, Circulation Res, 36, Suppl. 1, 223. Hayden, J.F., L.R. Johnson and R.P. Maickel, 1966, Construction and implantation of a permanent cannula for making injections into the lateral ventricle of the rat brain, Life Sci. 5, 1509. Ito, A. and S.M. Schanberg, 1974, Maintenance of tonic vasomotor activity by alpha and beta adrenergic mechanisms in medullary cardiovascular centers, J. Pharmacol. Exptl. Therap. 189,392. Laverty, R., 1962, Relation in rat hind-limb blood vessels between nervous vasomotor tone and the response to vasoconstrictor drugs, Brit. J. Pharmacol. 18,451. Laverty, R., 1973, The mechanisms of action of some antihypertensive drugs, Brit. Med. Bull. 29, 152. McCubbin, J.W., Y. Kaneko and I.H. Page, 1960, Ability of serotonin and norepinephrine to mimic the central effects of reserpine on vasomotor activity, Circulation Res. 8, 849. Nashold, B.S., E. Mannarino and M. Wunderlich,
J.M. ELLIOTT, D.W.J. CLARK 1962, Pressor-depressor blood pressure responses in the cat after intraventricular injection of drugs, Nature, London 193, 1297. Pichler, L. and W. Kobinger, 1976, Decrease of preganglionic sympathetic discharges by intracisternally administered ~-adrenergic imidazoline compounds, Naunyn-Schmiedeb. Arch. Pharmacol. 293, R16. Schmitt, H., and H. Schmitt, 1969, Localisation of the hypertensive effect of 2o(2-6-dichlorophenylamino)-2-imidazoline hydrochloride (St 155, Catapresan), European J. Pharmacol. 6, 8. Scriabine, A., B.V. Clineschmidt and C.S. Sweet, 1976, Central noradrenergic control of blood pressure, Ann. Rev. Pharmacol. Toxicol. 16, 113. Share, N.N: and K.I. Melville, 1963, Centrally mediated sympathetic cardiovascular responses induced by intraventricular norepinephrine, J. Pharmacol. Exptl. Therap. 141, 15. Smookler, H.H., W.B. Severs, W.J. Kinnard and J.P. Buckley, 1966, Centrally mediated cardiovascular effects of angiotensin II, J. Pharmacol. Exptl. Therap. 153, 485. Struyker Boudier, H.A.J., G. Smeets, G. Brouwer and J.M. Van Rossum, 1974, Central and peripheral alpha adrenergic activity of imidazoline derivatives, Life Sci. 15,887. Van Zwieten, P.A., 1973, The central action of antihypertensive drugs mediated via central ~-receptors, J. Pharm. Pharmacol. 25, 89.
