Gen. Pharmac. Vol. 23, No. 4, pp. 775-780, 1992 Printed in Great Britain. All rights reserved
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Lid
VASOMOTOR RESPONSES IN THE ISOLATED PERFUSED EXTERNAL A N D INTERNAL CAROTID VASCULAR BEDS OF THE RAT Jos[ V. VLSQUEZ~ and GIANNI PINARDI2. t Department of Physiopathology, Vargas School of Medicine, Faculty of Medicine and 2Department of Physiology, Faculty of Pharmacy, Central University of Venezuela, Caracas, Venezuela (Received 7 October 1991) Almract--l. The external (ECB) or the internal (ICB) carotid vascular beds of the rat were isolated and perfused with Krebs-Henseleit solution at constant flow (I ml/min). Changes in perfusion pressure (laP) were recorded after cervical sympathetic stimulation and after the administration of norepinephrine (NE) and serotonin (5-HT). 2. Sympathetic stimulation induced an increase in PP (vasoconstriction) in both vascular beds, however, this effect was significantly higher in the ECB than in the ICB. 3. Exogenous NE also induced a significantly higher contractile response in the ECB. 4. Prazosin (10 -s M) significantly inhibited the response to sympathetic stimulation and to NE both in the ECB and in the ICB, but yohimbine (10 -7 M) had no effect, suggesting that the vasoconstriction was mainly due to the activation of ~-adrenoceptors. 5. 5-HT induced a contractile response both in the ECB and the ICB. In contrast with the response to NE, the contraction induced by 5-HT in the ICB was significantly higher than in the ECB. 6. Ketanserine (10 -s M) antagonised both responses, indicating the involvement of 5-HT 2 receptors. 7. The contractile effect of 5-HT in the ECB was significantly enhanced by a subthreshold sympathetic stimulation that did not modify the PP by itself. This effect was not seen in the ICB. 8. The differential perfusions of the ECB or the ICB demonstrated a different reactivity of ECB and ICB, both to sympathetic stimulation and to the administration of exogenous NE or 5-HT. 9. Furthermore, the response to 5-HT in the ECB was modulated by a subthreshold sympathetic stimulation. 10. The results suggest that the different reactivity of the ECB and the ICB may be due to differences in the characteristics and/or density of adrenoceptors and 5-HT receptors in both vascular beds.
The present work was carried out with a methodology based on the differential perfusion of external (ECB) and internal (ICB) carotid vascular beds of isolated rat heads that allowed the recording of vasoconstrictor responses elicited either by sympathetic nerve stimulation or by the injection of exogenous vasoactive substances.
INTRODUCTION
Several authors have tried to assess cerebro-vascular reactivity by direct flowmeter measurements of cerebral blood flow (Liuch et al., 1975), by direct observation of pial vessels responses (Wahl et al., 1970) or by perfused isolated brain techniques (Rapela et al., 1967) in different animal species. However, these intents have not been entirely satisfactory. The responses of the cerebro-vascular bed to vasoactive substances have generally been inferred from studies utilising portions of cerebral arteries from rabbits, cats, dogs and monkeys (Duckles and Bevan, 1976; Edvinsson and Hardebo, 1976; Fu and Toda, 1983; Hayashi et al., 1985). The abovementioned studies rely on the behaviour of isolated major cerebral blood vessels from relatively large and expensive animals. An interesting technique, which can give useful information on v a s o m o t o r responses o f the cerebral vessels, was developed by Key et al. 0983), based on the in situ perfusion of the rat cerebral circulation.
MATERIALS AND METHODS
Spragne-Dawley rats of either sex (300-350g) were anaesthetised with i.p. sodium pcntobarbital (35 mg/kg) and prepared for the differential peffusion of either the external or the internal carotid vascular beds by removing the skin of the central aspect of the neck and inserting a cannula in the trachea to ensure a permeable airway. The right cervical sympathetic trunk was carefully dissected and cut, and the region of the right common carotid artery was exposed. Perfusion of the ECB To pcrfuse the right ECB, the left common carotid artery was tied off. The right internal carotid artery was traced to the base of the skull, the occipital artery was identified and both arteries were ligated. The right common carotid artery was cannulated with a polyethylene cannula (PE 50) and 500 units of heparin were slowly injected through the cannula to eliminate most of the blood within the vascular bed. The animal was killed by quickly opening the thorax and occluding the base of the aorta with a clamp. The head was
*To whom all correspondence should be addressed at present address: Department of Pharmacology, Faculty of Medicine, University of Chile, P.O. Box 70.000, Santiago 7, Chile. 775
776
Josl~ V. VLSQt~Z and GI^NN1 I~NAgDI
severed and placed in a perspex container. Perfusion was started with an LKB peristaltic flow inducer at a constant flow of I ml/min, using a modified Krebs--Henseleit solution of the following composition (mM): NaCI, 122.0; KCI, 4.7; CaCI 2, 2.0; MgCI 2, 1.2; KH2PO,, 1.2; NaHCO 3, 15.0; and glucose, 11.5. In order to increase the colloidal osmotic pressure, polyvinylpyrrolidone (40,000 m.w.) was added to the perfusate to produce a 4*/0 concentration. The perfusate was maintained at 37°C and bubbled with a 95% O5 and 5% CO2 gas mixture. The pH of the gasified solution was 7.,1. The mean perfusion pressure (PP) was recorded with a strain-gauge pressure transducer (LPU-0.1, TMI, Tokyo, Japan) in a Nihon-Kohden direct-writing polygraph under suitable amplification.
Table 1. Effectsof prazosin (10-s M) and yohimbine (10-7 M) on the increase in perfusion pressure (PP) induced by sympathetic stimulation in the external and internal carotid vascular beds of the rat PP increment (ram Hg) Control Prazosin Yohimbin¢ External carotid 29.6 + 2.4 14.8 ± 2.5* 25.8 + 4.6 (n = 23) Internal carotid 6.0 ± 0.6t 6.2 ± 0.4 5.8 + 1.4 (n = 24) Values are mean + SE. *P < 0.001 between control and prazosin. tP < 0.001 between control values.
Perfusion of the 1CB The right ICB was isolated by tying off the left common carotid, the right external carotid and the right pterygopalatine arteries. In addition, the vertebral arteries were cauterised at the level of the first cervical vertebra. In this way, most of the blood flow from the internal carotid circuit that could drain into extracranial vessels was occluded (Key et al., 1983). The skin overlying the dorsal surface of the skull was excised and a trephine was used to prepare a wide window over the parietal bone, which was later removed to make an incision on the confluence of the sagittal and transverse sinuses to provide a venous outlet for the perfusate. The parietal window also allowed for a certain expansion of the brain under small degrees of edema formation during the perfusion period, impeding extravascular compression on cerebral blood vessels that could modify the basal perfusion pressure. The right common carotid was cannulated, heparin was injected and then the rat was killed. The head was isolated and the perfusion was started as described for the ECB. To assess the degree of contamination of the ICB with blood of extracranial origin, in some experiments a 10% trypan blue solution was perfused into the ICB or the ECB for 15 rnin. Afterwards, the brain was removed, sectioned and photographed to observe the distribution of the dye. With ICB perfusion, the dye was distributed preferentially along the vascular beds of the right anterior and middle cerebral arteries. The cerebellar arteries were not involved in the perfusion. The contamination of these territories with ECB blood was negligible, as shown by the distribution of dye during ECB perfusion.
RESULTS
Effects of sympathetic stimulation The basal level o f PP in the ECB was 54.6 + 2.8 m m Hg. This value was significantly different from the value o b t a i n e d in the ICB at the same rate o f flow, which was 68.7 + 2.0 m m Hg. The stimulation o f the cervical sympathetic nerve induced a n increase o f 29.6 + 2.4 m m Hg in the ECB. In contrast, the response o f the ICB was significantly lower (6.0 ± 0.6 m m Hg; T a b l e 1). Table I shows t h a t prazosin, a selective ~ - a d r e n o ceptor a n t a g o n i s t , p r o d u c e d a significant reduction in the PP increase induced by sympathetic stimulation in the ECB, w i t h o u t affecting the response o f the ICB. Y o h i m b i n e , a selective a2-adrenoceptor antagonist, did n o t significantly modify the responses to s y m p a t h e t i c s t i m u l a t i o n in either beds.
Effects of norepinephrine (NE) N E administered in bolus injections of 1, 3 a n d 10/zg induced a d o s e - d e p e n d e n t increase in P P b o t h in E C B a n d ICB. However, the response observed in the ICB was significantly lower at all doses used (Fig. 1).
80
Experimental procedure After a 15-20min stabilisation period, the sectioned cervical sympathetic trunk was stimulated preganglionically in a cephalic direction with rectangular pulses (20 V, 20 Hz, 1.5 msec) delivered by a Grass S-44 stimulator to bipolar platinum electrodes for 15 sec. Several control stimulations were recorded. Increasing doses of drugs (norepinephrine or serotonin) were injected in the proximity of the carotid cannula in vols of 10-30/H with a Hamilton syringe. Injections were made after the increase in PP induced by the preceding dose reached basal levels. Antagonists [prazosin (10 -s M), yohimbine (10-TM)] and ketanserine (10 -7 M) were added to the Krebs-Henseleit solution and allowed to act for at least 20 min before repeating the drug injections. Under conditions of constant flow perfusion, any change in PP reflects a corresponding variation in vessel caliber. Changes in PP were expressed in mm Hg increase over basal level. Statistical analysis of the data was carried out using mean values -1- SEM. The significance was assessed by Student's t-test for paired and unpaired data. Differences were considered significant at P < 0.05. All drugs used in this work were obtained from Sigma Chemical (St Louis, Mo.), except prazosin and ketanserine which were kindly donated by Pfizer Laboratories and Jenssen Pharmaceuticals, respectively. Drugs were freshly dissolved in Krebs--Henseleit solution before each experiment.
//
1
2O
0-
I
3
10
NE/~g Fig. I. The effect of N E on the increase in PP in the E C B (0) and in the ICB (O) of the rat perfused with KrebsHense]eit solution at constant flow (1 ml/min). Values are mean :t: SEM of 15 experiments. All points are significantly different.
777
Vasomotor responses in carotid vascular beds 60
(A)
IS
I
0
40
20
Ub
-IE
E
I
3
1o
1
o
3
10
E U e-
eo-
2o -
(C)
1
(D)
Q. 13. 60-
15
4O
t0
-
20
/ o
1
3
0
lO
1
I
I
1
3
IO
NE/zg
Fig. 2. The effects of =-adrenoceptor antagonists on the increase in PP induced by NE in carotid vascular beds of the rat perfused with Krebs-Henseleit solution at constant flow (1 ml/min). (A) Effect of prazosin in the ECB. (B) Effect of prazosin in the ICB. (C) Effect of yohimbine in the ECB. (D) Effect of yohimbine in the ICB. Solid lines are control curves (O). Interrupted lines are prazosin (©) and yohimbine (rq) curves. Values are mean ± SEM of at least 10 experiments. Points of(C) and of(D) are not significantly different.
Figure 2 shows the effects of prazosin and yohimbine on the NE responses of the ECB and ICB. Prazosin (10-SM) significantly reduced the PP increase induced by NE in both vascular beds [Fig. 2(A) and (B)]; yohimbine (10 -7 M) did not significantly affect the responses [Fig. 2(C) and (D)].
Effects of serotonin (5-HT) 5-HT injected in doses of 0.03, 0.1, 0.3 and 1.0pg contracted both the ECB and ICB. The response of the ICB was significantly higher with all doses (Fig. 3). Ketanserine, a selective antagonist of 5-HT2 receptors, at a dose of l0 -7 M, significantly reduced the response to 5-HT in both vascular beds [Fig. 4(A) and (B)]. To study the possible interactions between sympathetic stimulation and activation of 5-HT receptors, the cervical sympathetic nerve was continuously stimulated, beginning 2 rain before the injection of 5-HT and maintaining the stimulation until the response was completed. A subthreshold stimulation (4V, 5 I-Iz, 1.5 mse¢) which alone did not produce increases in the PP of the vascular beds was used.
2O
15
| e
10
R5
o-
I O03
I 0.1
1 O.3
J 10
{5-HT/~
Fig. 3. The effect of 5-HT on the increase m PP m the ECB (Q) and in the ICB ( 0 ) of the rat perfused with Krebs-
Henseleit solution at constant flow (1 ral/min). Values are mean ± SEM of 12 experiments. All points are significantly different.
778
Jost V. V,~UEZ and GtANm PINARDI
L
(AI 13--
(B)
O~
T E E
._
10
S-
I1. 13.
0.03
0.t
0.3
I
0
0.03
0.1
0. 3
5 - H T/zg
Fig. 4. The effect of ketanserine on the increase in PP induced by 5-HT in carotid vascular beds of the rat perfused with Krebs-Henseleit solution at constant flow (I ml/min). (A) Effect of ketanserine in the ECB. (B) Effect of ketanserine in the ICB. Values are mean + SEM of at least 8 experiments. Control (O); ketanserine ((3). All points are significantly different.
Figure 5 shows that the effect of 5-HT was significantly increased in the ECB, but the subthreshold stimulation was not able to induce changes in the ICB, as can be seen in Fig. 5(A) and (B), respectively. D~CU~ION The methodology used in the present study provides evidence that this procedure is useful to compare the differential responses of ECB and ICB to exogenous vasoactive substances and sympathetic stimulation under conditions of flow. The dose-dependent vasoconstriction induced in both vascular beds by NE and
t5
(A)
5-HT was different in magnitude; this finding agrees with the accepted notion that the vasoconstrictor response of the cerebral arteries to 5-HT is greater than to NE (Hardebo et al., 1978; Toda, 1983), in contrast with peripheral arteries, which are more responsive to NE (Toda and Fujita, 1973). The response of the ICB to cervical sympathetic stimulation was significantly less than the response of the ECB. Essentially the same differential response was obtained with the administration of exogenous NE. Even though adrenergic sympathetic fibres are widely distributed in cerebral vessels (Edvinsson et al., 1972), previous reports clearly indicate that its
15
O=
/
10 O
E O r-
/:
3 n n
0.03
0.1
0.3 5-HT
0
I
I
I
0.03
0.1
0.3
~g
Fig. 5. The effect of a sustained subthreshold stimulation of the cervical sympathetic trunk on the increase in PP induced by 5-HT in carotid vascular beds of the rat perfused with Krebs-Henseleit solution at constant flow (1 ml/min). A subthreshold stimulation of 4 V, 5 Hz and 1.5 msec, which alone did not induce any increase in basal perfnsion pressure was started 2 min before the injection of serotonin and was maintained for the duration of the response. (A) Effect of subthreshold sympathetic stimulation in the ECB. (B) Effect of subthreshold sympathetic stimulation in the ICB. Values are mean + SEM of at least 8 experiments. Control (O); subthreshold sympathetic stimulation ((3). Points on (B) are not significantly different.
Vasomotor responses in carotid vascular beds ability to reduce cerebral blood flow by vasoconstriction is rather limited (Edvinsson and MacKenzie, 1976). Postsynaptic acadrenoceptors have been demonstrated in isolated cerebral arteries (Hayashi et al., 1985), but they seem to be different from those present in peripheral arteries in relation to their agonists and antagonists affinities (Edvinsson and Owman, 1974). Prazosin 0 0 -8 M) displayed a similar antagonistic action against the effect of exogenous NE in both vascular beds but surprisingly, the effect of sympathetic stimulation was antagonised only in the ECB. The ~l-adrenergic receptor stimulated by exogenous NE has probably similar pharmacological characteristics in the ECB and the ICB, since Hayashi et al. (1985) reported similar prazosin pA2 values for the isolated baboon middle cerebral artery (9.7) and mesenteric artery (8.95). However, the postsynaptic adrenoceptor activated by NE released from nerve terminals during sympathetic stimulation is almost completely blocked by prazosin in the ECB, while this apparently same type of receptor is unaffected by an equal dose of the antagonist in the ICB. There is no obvious explanation for this observation; one could postulate that prazosin, when perfused into the arterial lumen, cannot easily diffuse into the synaptic cleft of the sympathetic terminal in cerebral arteries with the same facility than in extracranial or peripheral arteries. Intraparenchymal and pial arterioles possess a continuous sheet of endothelial cells joined together by tight junctions, which is a singular property of the cerebral circulation (Fein et al., 1964; Westergaard and Brightman, 1973). This characteristic feature contributes to the morphological blood-brain barrier and may impair diffusion of substances from the lumen in a more pronounced way than in peripheral vessels. Nevertheless, Lee et al. (1976) using transmural nerve stimulation of rings of isolated rabbit basilar artery, found that phenoxybenzamine, phentolamine and tolazoline (a-adrenoceptor antagonists) in doses that abolished contractions to exogenous NE, potentiated and prolonged the contractile response to nerve stimulation. Our findings seem to support the idea that the adrenergic receptors of cerebral arteries differ from those of other arteries with respect to their sensitivity to neurotransmitters and their affinity constants for agonists and antagonists (Toda, 1983; Medgett and Langer, 1983). Although postsynaptic ct2-adrenoceptors have been demonstrated in isolated cerebral arteries (Toda, 1983; Sakakibara et al., 1982), in the present study yohimbine (10-7M) was not able to significantly affect the response to NE or to sympathetic stimulation in the ECB and the ICB. Probably, the dose of yohimbine was not high enough to block the a2-adrenoceptors. However, since prazosin was able to antagonise the responses to NE in both vascular beds at a concentration of l0 -8 M, these results suggest a different population of postsynaptic ~ - and a2-adrenoceptors. Serotonin induced a dose-dependent contractile response in both vascular beds, with significantly higher values in the ICB, in agreement with previous reports (Toda and Fujita, 1973; Edvinsson and Hardebo, 1976; Edvinsson et al., 1984). The results obtained with ketanserine seem to indicate that the
779
receptors involved in the response to 5-HT are of the 5-HT2 subtype, which is the receptor responsible for the contraction of vascular smooth muscle (Bradley et al., 1986). The response to 5-HT in the ECB was potentiated by a sustained subthreshold sympathetic stimulation. Small concentrations of NE are presumably released into the synaptic cleft by this stimulation and may produce a small increase in the intracellular Ca 2+ concentration by activating IPrlinked :t~-adrenoceptors in the vascular smooth muscle cells, increasing the sensitivity to other vasoconstrictors. Several reports indicate that 5-HT may interact with the adrenergic system in isolated vessels and perfused vascular beds (Wilton and McCalden, 1977; Van Nueten et al., 1982). In general, the interaction consists of a potentiation of the adrenergic vasoconstrictor response in peripheral vessels. Our results indicate that the adrenergic system can also potentiate serotonergic responses. The ICB did not show the same behaviour, a fact which could be related to the difference in the receptor sensitivity and/or density of this vascular bed. In conclusion, the differential perfusion method described allows the recording of vasomotor responses in the ECB and ICB. The results suggest that the ICB is less responsive than ECB to NE and sympathetic stimulation, but 5-HT is a more potent vasoconstrictor in this vascular bed. Acknowledgements--This work was supported in part by Project S 1-1062, CONICIT, Venezuela and Project F-06.1.84, CDCH, Central University of Venezuela. The authors are grateful to Dr H. F. Miranda for helpful suggestions concerning the manuscript. REFERENCES
Bradley P. B., Engel G., Feniuk W., Fozard J. R., Humphrey P. P. A., Middlerniss D. N., Mylecharane E. J., Richardson B. P. and Saxena P. R. (1986) Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine. Neuropharmacology 25, 563-576. Duckies S. P. and Bevan J. A. (1976) Pharmacological characterization of adrenergic receptors of rabbit cerebral artery in vitro. J. Pharmac. exp. Ther. 197, 371-378. Edvinsson L. and Hardebo J. A. (1976) Characterization of serotonin-receptors in intracranial and extracranial vessels. Acta physiol, scand. 97, 523-525. Edvinsson L. and MacKenzie E. T. (1976) Amine mechanisms in the cerebral circulation. Pharmac. Rev. 28, 275-348. Edvinsson L. and Owman Ch. (1974) Pharmacological characterization of adrenergic alpha and beta receptors mediating the vasomotor responses of cerebral arteries in vitro. Circulation Res. 35, 835449. Edvinsson L., Nielsen K. C., Owman Ch. and Sporrong B. L. (1972) Cholinergic mechanisms in pial vessels. Histc,zhemistry, electron microscopy and pharmacology. Z. Zellforsch. mikrosk. Anat. 134, 311-325. Edvinsson L., Birath E., Uddman R., Lee T. J-F., Duverger D., MacKenzie E. T. and Scatton B. (1984) Indolaminergic mechanism in brain vessels, localization, concentration, uptake and in vitro responses of 5-hydroxytryptamine. Acta physiol, scand. 121, 291-299. Fein J. M., Flor W. J., Cohan S. L. and Parkhurst J. (1974) Sequential changes of vascular ultrastructure in experimental cerebral vasospasm. J. Neurosurg. 41, 49-58.
780
Jo$1~ V. VASQUEZand GIANNIPINARDI
Fu L. H. W. and Toda N. (1983) Analysis of the contractile responses to serotonin and tryptamine of isolated dog cerebral, femoral and mesenteric arteries. ,lap. J. Pharmac. 33, 473-480. Hardebo J. E., Edvinsson L., Owman Ch. and Svendgaard N. A. A. (1978) Potentiation and antagonism of serotonin effects on intracranial and extracranial vessels. Neurology 28, 64-70. Hayashi S., Park M. K. and Kuehl T. J. (1985) Post-synaptic alpha adrenoceptors in baboon cerebral and mesenteric arteries. J. Pharmac. exp. Ther. 735, 113-121. Key B. J., Ruddock F. S. and Harris P. (1983) Vasomotor responses recorded using the in situ perfusion of a portion of the rat cerebral circulation. J. Pharmac. Meth. 9, 201-208. Lee T. J-F., Su C. and Bevan J. A. (1976) Neurogenic sympathetic vasoconstriction of the rabbit basilar artery. Circulation Res. 39, 120-126. Lluch S., Gomez B., Alborch E. and Urquilla P. R. (1975) Adrenergic mechanisms in cerebral circulation of the goat. Am. J. Physiol. 228, 985-989. Medgett I. C. and Langer S. Z. (1983) Characterization of smooth muscle alpha-adrenoceptors and of responses to electrical stimulation in the cat isolated perfused middle cerebral artery. Naunyn-Schmiedebergs Arch. Pharmac. 373, 24-32. Rapela C. E., Green H. D. and Dcnison A. B. Jr (1967) Baroreceptor reflexes and autoregulation of cerebral blood flow in the dog. Circulation Res. 21, 559-568.
Sakakibara Y., Fujiwara M. and Muramatsu 1. (1982) Pharmacological characterization of the alpha-adrenoceptors of the dog basilar artery. Naunyn-Schmiedebergs Arch. Pharmac. 319, 1-7. Toda N. (1983) Alpha-adrenergic receptor subtypes in human, monkey and dog cerebral arteries. J. Pharmac. exp. Ther. 226, 861-868. Toda N. and Fujita Y. (1973) Responsiveness of isolated cerebral and peripheral arteries to serotonin, norepinephfine, and transmural electrical stimulation. Circulation Res. 33, 98-104. Van Nueten J. M., Hanssen P. A. J., DeRidder W. and Vanhoutte P. M. (1982) Interaction between 5-hydroxytryptamine and other vasoconstrictor substances in the isolated artery of the rabbit; effect of ketanserine (R 41 468). Eur. J. Pharmac. 77, 281-287. Wahl M., Deetjen P., Thurau K., Ingvar D. H. and Lassen N. A. (1970) Micropuncture evaluation of the importance of perivascular pH for the arteriolar diameter on the brain surface. Pfliigers Arch. ges. Physiol. 316, 152-163. Westergaard E. and Brightman M. W. (1973) Transport of proteins across normal cerebral arterioles. J. comp. Neurol. 152, 17-44. Wilton P. B. and McCalden T. A. (1977) The interaction between noradrenaline and 5-hydroxytryptamine in the isolated perfused rat hind limb. Eur. J. Pharmac. 46, 213-219.
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Lid
VASOMOTOR RESPONSES IN THE ISOLATED PERFUSED EXTERNAL A N D INTERNAL CAROTID VASCULAR BEDS OF THE RAT Jos[ V. VLSQUEZ~ and GIANNI PINARDI2. t Department of Physiopathology, Vargas School of Medicine, Faculty of Medicine and 2Department of Physiology, Faculty of Pharmacy, Central University of Venezuela, Caracas, Venezuela (Received 7 October 1991) Almract--l. The external (ECB) or the internal (ICB) carotid vascular beds of the rat were isolated and perfused with Krebs-Henseleit solution at constant flow (I ml/min). Changes in perfusion pressure (laP) were recorded after cervical sympathetic stimulation and after the administration of norepinephrine (NE) and serotonin (5-HT). 2. Sympathetic stimulation induced an increase in PP (vasoconstriction) in both vascular beds, however, this effect was significantly higher in the ECB than in the ICB. 3. Exogenous NE also induced a significantly higher contractile response in the ECB. 4. Prazosin (10 -s M) significantly inhibited the response to sympathetic stimulation and to NE both in the ECB and in the ICB, but yohimbine (10 -7 M) had no effect, suggesting that the vasoconstriction was mainly due to the activation of ~-adrenoceptors. 5. 5-HT induced a contractile response both in the ECB and the ICB. In contrast with the response to NE, the contraction induced by 5-HT in the ICB was significantly higher than in the ECB. 6. Ketanserine (10 -s M) antagonised both responses, indicating the involvement of 5-HT 2 receptors. 7. The contractile effect of 5-HT in the ECB was significantly enhanced by a subthreshold sympathetic stimulation that did not modify the PP by itself. This effect was not seen in the ICB. 8. The differential perfusions of the ECB or the ICB demonstrated a different reactivity of ECB and ICB, both to sympathetic stimulation and to the administration of exogenous NE or 5-HT. 9. Furthermore, the response to 5-HT in the ECB was modulated by a subthreshold sympathetic stimulation. 10. The results suggest that the different reactivity of the ECB and the ICB may be due to differences in the characteristics and/or density of adrenoceptors and 5-HT receptors in both vascular beds.
The present work was carried out with a methodology based on the differential perfusion of external (ECB) and internal (ICB) carotid vascular beds of isolated rat heads that allowed the recording of vasoconstrictor responses elicited either by sympathetic nerve stimulation or by the injection of exogenous vasoactive substances.
INTRODUCTION
Several authors have tried to assess cerebro-vascular reactivity by direct flowmeter measurements of cerebral blood flow (Liuch et al., 1975), by direct observation of pial vessels responses (Wahl et al., 1970) or by perfused isolated brain techniques (Rapela et al., 1967) in different animal species. However, these intents have not been entirely satisfactory. The responses of the cerebro-vascular bed to vasoactive substances have generally been inferred from studies utilising portions of cerebral arteries from rabbits, cats, dogs and monkeys (Duckles and Bevan, 1976; Edvinsson and Hardebo, 1976; Fu and Toda, 1983; Hayashi et al., 1985). The abovementioned studies rely on the behaviour of isolated major cerebral blood vessels from relatively large and expensive animals. An interesting technique, which can give useful information on v a s o m o t o r responses o f the cerebral vessels, was developed by Key et al. 0983), based on the in situ perfusion of the rat cerebral circulation.
MATERIALS AND METHODS
Spragne-Dawley rats of either sex (300-350g) were anaesthetised with i.p. sodium pcntobarbital (35 mg/kg) and prepared for the differential peffusion of either the external or the internal carotid vascular beds by removing the skin of the central aspect of the neck and inserting a cannula in the trachea to ensure a permeable airway. The right cervical sympathetic trunk was carefully dissected and cut, and the region of the right common carotid artery was exposed. Perfusion of the ECB To pcrfuse the right ECB, the left common carotid artery was tied off. The right internal carotid artery was traced to the base of the skull, the occipital artery was identified and both arteries were ligated. The right common carotid artery was cannulated with a polyethylene cannula (PE 50) and 500 units of heparin were slowly injected through the cannula to eliminate most of the blood within the vascular bed. The animal was killed by quickly opening the thorax and occluding the base of the aorta with a clamp. The head was
*To whom all correspondence should be addressed at present address: Department of Pharmacology, Faculty of Medicine, University of Chile, P.O. Box 70.000, Santiago 7, Chile. 775
776
Josl~ V. VLSQt~Z and GI^NN1 I~NAgDI
severed and placed in a perspex container. Perfusion was started with an LKB peristaltic flow inducer at a constant flow of I ml/min, using a modified Krebs--Henseleit solution of the following composition (mM): NaCI, 122.0; KCI, 4.7; CaCI 2, 2.0; MgCI 2, 1.2; KH2PO,, 1.2; NaHCO 3, 15.0; and glucose, 11.5. In order to increase the colloidal osmotic pressure, polyvinylpyrrolidone (40,000 m.w.) was added to the perfusate to produce a 4*/0 concentration. The perfusate was maintained at 37°C and bubbled with a 95% O5 and 5% CO2 gas mixture. The pH of the gasified solution was 7.,1. The mean perfusion pressure (PP) was recorded with a strain-gauge pressure transducer (LPU-0.1, TMI, Tokyo, Japan) in a Nihon-Kohden direct-writing polygraph under suitable amplification.
Table 1. Effectsof prazosin (10-s M) and yohimbine (10-7 M) on the increase in perfusion pressure (PP) induced by sympathetic stimulation in the external and internal carotid vascular beds of the rat PP increment (ram Hg) Control Prazosin Yohimbin¢ External carotid 29.6 + 2.4 14.8 ± 2.5* 25.8 + 4.6 (n = 23) Internal carotid 6.0 ± 0.6t 6.2 ± 0.4 5.8 + 1.4 (n = 24) Values are mean + SE. *P < 0.001 between control and prazosin. tP < 0.001 between control values.
Perfusion of the 1CB The right ICB was isolated by tying off the left common carotid, the right external carotid and the right pterygopalatine arteries. In addition, the vertebral arteries were cauterised at the level of the first cervical vertebra. In this way, most of the blood flow from the internal carotid circuit that could drain into extracranial vessels was occluded (Key et al., 1983). The skin overlying the dorsal surface of the skull was excised and a trephine was used to prepare a wide window over the parietal bone, which was later removed to make an incision on the confluence of the sagittal and transverse sinuses to provide a venous outlet for the perfusate. The parietal window also allowed for a certain expansion of the brain under small degrees of edema formation during the perfusion period, impeding extravascular compression on cerebral blood vessels that could modify the basal perfusion pressure. The right common carotid was cannulated, heparin was injected and then the rat was killed. The head was isolated and the perfusion was started as described for the ECB. To assess the degree of contamination of the ICB with blood of extracranial origin, in some experiments a 10% trypan blue solution was perfused into the ICB or the ECB for 15 rnin. Afterwards, the brain was removed, sectioned and photographed to observe the distribution of the dye. With ICB perfusion, the dye was distributed preferentially along the vascular beds of the right anterior and middle cerebral arteries. The cerebellar arteries were not involved in the perfusion. The contamination of these territories with ECB blood was negligible, as shown by the distribution of dye during ECB perfusion.
RESULTS
Effects of sympathetic stimulation The basal level o f PP in the ECB was 54.6 + 2.8 m m Hg. This value was significantly different from the value o b t a i n e d in the ICB at the same rate o f flow, which was 68.7 + 2.0 m m Hg. The stimulation o f the cervical sympathetic nerve induced a n increase o f 29.6 + 2.4 m m Hg in the ECB. In contrast, the response o f the ICB was significantly lower (6.0 ± 0.6 m m Hg; T a b l e 1). Table I shows t h a t prazosin, a selective ~ - a d r e n o ceptor a n t a g o n i s t , p r o d u c e d a significant reduction in the PP increase induced by sympathetic stimulation in the ECB, w i t h o u t affecting the response o f the ICB. Y o h i m b i n e , a selective a2-adrenoceptor antagonist, did n o t significantly modify the responses to s y m p a t h e t i c s t i m u l a t i o n in either beds.
Effects of norepinephrine (NE) N E administered in bolus injections of 1, 3 a n d 10/zg induced a d o s e - d e p e n d e n t increase in P P b o t h in E C B a n d ICB. However, the response observed in the ICB was significantly lower at all doses used (Fig. 1).
80
Experimental procedure After a 15-20min stabilisation period, the sectioned cervical sympathetic trunk was stimulated preganglionically in a cephalic direction with rectangular pulses (20 V, 20 Hz, 1.5 msec) delivered by a Grass S-44 stimulator to bipolar platinum electrodes for 15 sec. Several control stimulations were recorded. Increasing doses of drugs (norepinephrine or serotonin) were injected in the proximity of the carotid cannula in vols of 10-30/H with a Hamilton syringe. Injections were made after the increase in PP induced by the preceding dose reached basal levels. Antagonists [prazosin (10 -s M), yohimbine (10-TM)] and ketanserine (10 -7 M) were added to the Krebs-Henseleit solution and allowed to act for at least 20 min before repeating the drug injections. Under conditions of constant flow perfusion, any change in PP reflects a corresponding variation in vessel caliber. Changes in PP were expressed in mm Hg increase over basal level. Statistical analysis of the data was carried out using mean values -1- SEM. The significance was assessed by Student's t-test for paired and unpaired data. Differences were considered significant at P < 0.05. All drugs used in this work were obtained from Sigma Chemical (St Louis, Mo.), except prazosin and ketanserine which were kindly donated by Pfizer Laboratories and Jenssen Pharmaceuticals, respectively. Drugs were freshly dissolved in Krebs--Henseleit solution before each experiment.
//
1
2O
0-
I
3
10
NE/~g Fig. I. The effect of N E on the increase in PP in the E C B (0) and in the ICB (O) of the rat perfused with KrebsHense]eit solution at constant flow (1 ml/min). Values are mean :t: SEM of 15 experiments. All points are significantly different.
777
Vasomotor responses in carotid vascular beds 60
(A)
IS
I
0
40
20
Ub
-IE
E
I
3
1o
1
o
3
10
E U e-
eo-
2o -
(C)
1
(D)
Q. 13. 60-
15
4O
t0
-
20
/ o
1
3
0
lO
1
I
I
1
3
IO
NE/zg
Fig. 2. The effects of =-adrenoceptor antagonists on the increase in PP induced by NE in carotid vascular beds of the rat perfused with Krebs-Henseleit solution at constant flow (1 ml/min). (A) Effect of prazosin in the ECB. (B) Effect of prazosin in the ICB. (C) Effect of yohimbine in the ECB. (D) Effect of yohimbine in the ICB. Solid lines are control curves (O). Interrupted lines are prazosin (©) and yohimbine (rq) curves. Values are mean ± SEM of at least 10 experiments. Points of(C) and of(D) are not significantly different.
Figure 2 shows the effects of prazosin and yohimbine on the NE responses of the ECB and ICB. Prazosin (10-SM) significantly reduced the PP increase induced by NE in both vascular beds [Fig. 2(A) and (B)]; yohimbine (10 -7 M) did not significantly affect the responses [Fig. 2(C) and (D)].
Effects of serotonin (5-HT) 5-HT injected in doses of 0.03, 0.1, 0.3 and 1.0pg contracted both the ECB and ICB. The response of the ICB was significantly higher with all doses (Fig. 3). Ketanserine, a selective antagonist of 5-HT2 receptors, at a dose of l0 -7 M, significantly reduced the response to 5-HT in both vascular beds [Fig. 4(A) and (B)]. To study the possible interactions between sympathetic stimulation and activation of 5-HT receptors, the cervical sympathetic nerve was continuously stimulated, beginning 2 rain before the injection of 5-HT and maintaining the stimulation until the response was completed. A subthreshold stimulation (4V, 5 I-Iz, 1.5 mse¢) which alone did not produce increases in the PP of the vascular beds was used.
2O
15
| e
10
R5
o-
I O03
I 0.1
1 O.3
J 10
{5-HT/~
Fig. 3. The effect of 5-HT on the increase m PP m the ECB (Q) and in the ICB ( 0 ) of the rat perfused with Krebs-
Henseleit solution at constant flow (1 ral/min). Values are mean ± SEM of 12 experiments. All points are significantly different.
778
Jost V. V,~UEZ and GtANm PINARDI
L
(AI 13--
(B)
O~
T E E
._
10
S-
I1. 13.
0.03
0.t
0.3
I
0
0.03
0.1
0. 3
5 - H T/zg
Fig. 4. The effect of ketanserine on the increase in PP induced by 5-HT in carotid vascular beds of the rat perfused with Krebs-Henseleit solution at constant flow (I ml/min). (A) Effect of ketanserine in the ECB. (B) Effect of ketanserine in the ICB. Values are mean + SEM of at least 8 experiments. Control (O); ketanserine ((3). All points are significantly different.
Figure 5 shows that the effect of 5-HT was significantly increased in the ECB, but the subthreshold stimulation was not able to induce changes in the ICB, as can be seen in Fig. 5(A) and (B), respectively. D~CU~ION The methodology used in the present study provides evidence that this procedure is useful to compare the differential responses of ECB and ICB to exogenous vasoactive substances and sympathetic stimulation under conditions of flow. The dose-dependent vasoconstriction induced in both vascular beds by NE and
t5
(A)
5-HT was different in magnitude; this finding agrees with the accepted notion that the vasoconstrictor response of the cerebral arteries to 5-HT is greater than to NE (Hardebo et al., 1978; Toda, 1983), in contrast with peripheral arteries, which are more responsive to NE (Toda and Fujita, 1973). The response of the ICB to cervical sympathetic stimulation was significantly less than the response of the ECB. Essentially the same differential response was obtained with the administration of exogenous NE. Even though adrenergic sympathetic fibres are widely distributed in cerebral vessels (Edvinsson et al., 1972), previous reports clearly indicate that its
15
O=
/
10 O
E O r-
/:
3 n n
0.03
0.1
0.3 5-HT
0
I
I
I
0.03
0.1
0.3
~g
Fig. 5. The effect of a sustained subthreshold stimulation of the cervical sympathetic trunk on the increase in PP induced by 5-HT in carotid vascular beds of the rat perfused with Krebs-Henseleit solution at constant flow (1 ml/min). A subthreshold stimulation of 4 V, 5 Hz and 1.5 msec, which alone did not induce any increase in basal perfnsion pressure was started 2 min before the injection of serotonin and was maintained for the duration of the response. (A) Effect of subthreshold sympathetic stimulation in the ECB. (B) Effect of subthreshold sympathetic stimulation in the ICB. Values are mean + SEM of at least 8 experiments. Control (O); subthreshold sympathetic stimulation ((3). Points on (B) are not significantly different.
Vasomotor responses in carotid vascular beds ability to reduce cerebral blood flow by vasoconstriction is rather limited (Edvinsson and MacKenzie, 1976). Postsynaptic acadrenoceptors have been demonstrated in isolated cerebral arteries (Hayashi et al., 1985), but they seem to be different from those present in peripheral arteries in relation to their agonists and antagonists affinities (Edvinsson and Owman, 1974). Prazosin 0 0 -8 M) displayed a similar antagonistic action against the effect of exogenous NE in both vascular beds but surprisingly, the effect of sympathetic stimulation was antagonised only in the ECB. The ~l-adrenergic receptor stimulated by exogenous NE has probably similar pharmacological characteristics in the ECB and the ICB, since Hayashi et al. (1985) reported similar prazosin pA2 values for the isolated baboon middle cerebral artery (9.7) and mesenteric artery (8.95). However, the postsynaptic adrenoceptor activated by NE released from nerve terminals during sympathetic stimulation is almost completely blocked by prazosin in the ECB, while this apparently same type of receptor is unaffected by an equal dose of the antagonist in the ICB. There is no obvious explanation for this observation; one could postulate that prazosin, when perfused into the arterial lumen, cannot easily diffuse into the synaptic cleft of the sympathetic terminal in cerebral arteries with the same facility than in extracranial or peripheral arteries. Intraparenchymal and pial arterioles possess a continuous sheet of endothelial cells joined together by tight junctions, which is a singular property of the cerebral circulation (Fein et al., 1964; Westergaard and Brightman, 1973). This characteristic feature contributes to the morphological blood-brain barrier and may impair diffusion of substances from the lumen in a more pronounced way than in peripheral vessels. Nevertheless, Lee et al. (1976) using transmural nerve stimulation of rings of isolated rabbit basilar artery, found that phenoxybenzamine, phentolamine and tolazoline (a-adrenoceptor antagonists) in doses that abolished contractions to exogenous NE, potentiated and prolonged the contractile response to nerve stimulation. Our findings seem to support the idea that the adrenergic receptors of cerebral arteries differ from those of other arteries with respect to their sensitivity to neurotransmitters and their affinity constants for agonists and antagonists (Toda, 1983; Medgett and Langer, 1983). Although postsynaptic ct2-adrenoceptors have been demonstrated in isolated cerebral arteries (Toda, 1983; Sakakibara et al., 1982), in the present study yohimbine (10-7M) was not able to significantly affect the response to NE or to sympathetic stimulation in the ECB and the ICB. Probably, the dose of yohimbine was not high enough to block the a2-adrenoceptors. However, since prazosin was able to antagonise the responses to NE in both vascular beds at a concentration of l0 -8 M, these results suggest a different population of postsynaptic ~ - and a2-adrenoceptors. Serotonin induced a dose-dependent contractile response in both vascular beds, with significantly higher values in the ICB, in agreement with previous reports (Toda and Fujita, 1973; Edvinsson and Hardebo, 1976; Edvinsson et al., 1984). The results obtained with ketanserine seem to indicate that the
779
receptors involved in the response to 5-HT are of the 5-HT2 subtype, which is the receptor responsible for the contraction of vascular smooth muscle (Bradley et al., 1986). The response to 5-HT in the ECB was potentiated by a sustained subthreshold sympathetic stimulation. Small concentrations of NE are presumably released into the synaptic cleft by this stimulation and may produce a small increase in the intracellular Ca 2+ concentration by activating IPrlinked :t~-adrenoceptors in the vascular smooth muscle cells, increasing the sensitivity to other vasoconstrictors. Several reports indicate that 5-HT may interact with the adrenergic system in isolated vessels and perfused vascular beds (Wilton and McCalden, 1977; Van Nueten et al., 1982). In general, the interaction consists of a potentiation of the adrenergic vasoconstrictor response in peripheral vessels. Our results indicate that the adrenergic system can also potentiate serotonergic responses. The ICB did not show the same behaviour, a fact which could be related to the difference in the receptor sensitivity and/or density of this vascular bed. In conclusion, the differential perfusion method described allows the recording of vasomotor responses in the ECB and ICB. The results suggest that the ICB is less responsive than ECB to NE and sympathetic stimulation, but 5-HT is a more potent vasoconstrictor in this vascular bed. Acknowledgements--This work was supported in part by Project S 1-1062, CONICIT, Venezuela and Project F-06.1.84, CDCH, Central University of Venezuela. The authors are grateful to Dr H. F. Miranda for helpful suggestions concerning the manuscript. REFERENCES
Bradley P. B., Engel G., Feniuk W., Fozard J. R., Humphrey P. P. A., Middlerniss D. N., Mylecharane E. J., Richardson B. P. and Saxena P. R. (1986) Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine. Neuropharmacology 25, 563-576. Duckies S. P. and Bevan J. A. (1976) Pharmacological characterization of adrenergic receptors of rabbit cerebral artery in vitro. J. Pharmac. exp. Ther. 197, 371-378. Edvinsson L. and Hardebo J. A. (1976) Characterization of serotonin-receptors in intracranial and extracranial vessels. Acta physiol, scand. 97, 523-525. Edvinsson L. and MacKenzie E. T. (1976) Amine mechanisms in the cerebral circulation. Pharmac. Rev. 28, 275-348. Edvinsson L. and Owman Ch. (1974) Pharmacological characterization of adrenergic alpha and beta receptors mediating the vasomotor responses of cerebral arteries in vitro. Circulation Res. 35, 835449. Edvinsson L., Nielsen K. C., Owman Ch. and Sporrong B. L. (1972) Cholinergic mechanisms in pial vessels. Histc,zhemistry, electron microscopy and pharmacology. Z. Zellforsch. mikrosk. Anat. 134, 311-325. Edvinsson L., Birath E., Uddman R., Lee T. J-F., Duverger D., MacKenzie E. T. and Scatton B. (1984) Indolaminergic mechanism in brain vessels, localization, concentration, uptake and in vitro responses of 5-hydroxytryptamine. Acta physiol, scand. 121, 291-299. Fein J. M., Flor W. J., Cohan S. L. and Parkhurst J. (1974) Sequential changes of vascular ultrastructure in experimental cerebral vasospasm. J. Neurosurg. 41, 49-58.
780
Jo$1~ V. VASQUEZand GIANNIPINARDI
Fu L. H. W. and Toda N. (1983) Analysis of the contractile responses to serotonin and tryptamine of isolated dog cerebral, femoral and mesenteric arteries. ,lap. J. Pharmac. 33, 473-480. Hardebo J. E., Edvinsson L., Owman Ch. and Svendgaard N. A. A. (1978) Potentiation and antagonism of serotonin effects on intracranial and extracranial vessels. Neurology 28, 64-70. Hayashi S., Park M. K. and Kuehl T. J. (1985) Post-synaptic alpha adrenoceptors in baboon cerebral and mesenteric arteries. J. Pharmac. exp. Ther. 735, 113-121. Key B. J., Ruddock F. S. and Harris P. (1983) Vasomotor responses recorded using the in situ perfusion of a portion of the rat cerebral circulation. J. Pharmac. Meth. 9, 201-208. Lee T. J-F., Su C. and Bevan J. A. (1976) Neurogenic sympathetic vasoconstriction of the rabbit basilar artery. Circulation Res. 39, 120-126. Lluch S., Gomez B., Alborch E. and Urquilla P. R. (1975) Adrenergic mechanisms in cerebral circulation of the goat. Am. J. Physiol. 228, 985-989. Medgett I. C. and Langer S. Z. (1983) Characterization of smooth muscle alpha-adrenoceptors and of responses to electrical stimulation in the cat isolated perfused middle cerebral artery. Naunyn-Schmiedebergs Arch. Pharmac. 373, 24-32. Rapela C. E., Green H. D. and Dcnison A. B. Jr (1967) Baroreceptor reflexes and autoregulation of cerebral blood flow in the dog. Circulation Res. 21, 559-568.
Sakakibara Y., Fujiwara M. and Muramatsu 1. (1982) Pharmacological characterization of the alpha-adrenoceptors of the dog basilar artery. Naunyn-Schmiedebergs Arch. Pharmac. 319, 1-7. Toda N. (1983) Alpha-adrenergic receptor subtypes in human, monkey and dog cerebral arteries. J. Pharmac. exp. Ther. 226, 861-868. Toda N. and Fujita Y. (1973) Responsiveness of isolated cerebral and peripheral arteries to serotonin, norepinephfine, and transmural electrical stimulation. Circulation Res. 33, 98-104. Van Nueten J. M., Hanssen P. A. J., DeRidder W. and Vanhoutte P. M. (1982) Interaction between 5-hydroxytryptamine and other vasoconstrictor substances in the isolated artery of the rabbit; effect of ketanserine (R 41 468). Eur. J. Pharmac. 77, 281-287. Wahl M., Deetjen P., Thurau K., Ingvar D. H. and Lassen N. A. (1970) Micropuncture evaluation of the importance of perivascular pH for the arteriolar diameter on the brain surface. Pfliigers Arch. ges. Physiol. 316, 152-163. Westergaard E. and Brightman M. W. (1973) Transport of proteins across normal cerebral arterioles. J. comp. Neurol. 152, 17-44. Wilton P. B. and McCalden T. A. (1977) The interaction between noradrenaline and 5-hydroxytryptamine in the isolated perfused rat hind limb. Eur. J. Pharmac. 46, 213-219.
