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Intrinsic and extrinsic mechanisms involved in the cerebrovascular reaction elicited by immobilization stress in rabbits
Brain Research, 340 (1985) 305-314
3(15
Elsevier BRE I0903
Intrinsic and Extrinsic Mechanisms Involved in the Cerebrovascular Reaction Elicited by Immobilization Stress in Rabbits E. PINARD, P. LACOMBE, A. M. REYNIER-REBUFFEL and J. SEYLAZ Laboratoire de Physiologie et Physiopathologie COrObrovasculaire, E. R.A. 361 C. N. R. S.. U. 182 l. N. S. E. R.M., Universit~ Paris VII, Paris (France
(Accepted November 6th, 1984) KCv words: cerebral blood flow - - cerebral metabolism - - pO 2- - pCO z - - immobilization stress - - neurogenic control
Variations in cerebral blood flow and partial pressures of oxygen and carbon dioxide (pO2, pCO2) were studied in rabbits during short-duration (1 min) immobilization stress. The techniques used enabled us to determine these variables locally in the caudate nucleus in a continuous, simultaneous and quantitative fashion. It could be shown that cerebral blood flow and arterial blood pressure increased in parallel immediately after inducing the stress reaction, and that pO 2 increased further, indicating that cerebral oxygen supply is maintained by the hyperaemia. Previous administration of a fl-receptor blocker or of a cholinergic receptor blocker significantly diminished the cerebrovascular reaction to stress, inducing a decrease in pO 2 during the reaction. Administration of both blockers nearly abolished the cerebral vasodilatation studied. Previous administration of an a-receptor blocker enhanced the reactive hyperaemia. No disturbance of the blood-brain barrier could be observed in rabbits subjected to stress. Intravenous injection of adrenaline, as well as angiotensin II inducing similar increases in blood pressure, had no comparable effect on the blood flow. The conclusion is that in this model of anxiety, neurogenic mechanisms are involved in the provision of a sufficient oxygen supply to the brain.
INTRODUCTION
stress reaction were still unknown since they had not been able to prove that catecholamines act on specif-
Cerebral circulation and metabolism are not only a
ic brain areas or on peripheral receptors that second-
subject of interest with regard to cerebrovascular dis-
arily influence neuronal activity. Contradictory re-
eases, but also in relation to critical physiological sit-
sults were obtained by O h at a et al. 27 working on re-
uations, In fact, more than 30 years ago, Kety 16 dem-
gional cerebral blood flow in the spontaneously
onstrated that anxiety in human beings can be asso-
breathing rat: they d e m o n s t r a t e d that the p~CO2 is
ciated with an increase in cerebral metabolic rate.
responsible for at most 20% of the variation of cere-
F u r t h e r m o r e , an increase in cerebral blood flow and
bral blood flow during immobilization stress. In view
oxygen consumption was observed during infusion of
of this controversy, the purpose of this study was to
epinephrine in a dose which raised arterial pressure
investigate the possible i n v o l v e m e n t of intrinsic and
and caused symptoms of anxiety17. M o r e recently,
extrinsic mechanisms in the variations of local cere-
Carlsson et al.4 d e v e l o p e d an experimental model of
bral circulation and metabolism during immobiliza-
immobilization stress in rats which provided evidence
tion stress. Accordingly we made continuous meas-
that stressful situations may cause significant increas-
urements on the time course of the p h e n o m e n o n and
es in global cerebral blood flow and metabolism. This
on the possible interference of various receptor
model enabled these workers to study the mechanisms of these increases and to d e t e r m i n e that they
blockers with the stress reaction. Several systemic and cerebrovascular variables were m o n i t o r e d simul-
were mediated via release of catecholamines from
taneously throughout experiments in which short-
the adrenal glands 5. H o w e v e r , they concluded that
lasting (1 min) immobilization stress reactions were
the
elicited periodically.
detailed
mechanisms
of the
cerebrovascular
Correspondence: E. Pinard, 10, avenue de Verdun, 75010 Paris, France.
0006-8993/85/$113.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
306 MATERIALS AND METHODS
Techniques Cerebral blood flow (CBF) was measured continuously in the caudate nucleus by means of a thermoclearance technique 37. This method is based on the thermal conductivity of the tissue which varies when blood flow varies. It requires the use of two heat-sensitive probes, one being heated to about one degree above the temperature of the second, unheated probe, used as a reference. A feed-back system maintains the temperature difference constant and the heating current maintaining this temperature difference is linearly related to the variations in blood flow. These variations can be expressed as per cent deviations about the baseline flow since at the end of the experiment, when the animal was killed with an overdose of i.v. pentobarbital, the zero level of cerebral blood flow was obtained. Response time of the blood flow measurement was 1-2 s. The thermoprobe consisted of a glass tube (0.7 m m o.d.) containing a thermistor, around which a heating coil was wound. Tissue partial pressures of oxygen and carbon dioxide (pO 2, pCO2) were quantitatively and continuously measured by mass spectrometry in the same nucleus. A detailed analysis of the theory and description of the technique used have already been published 39. Briefly, gases are withdrawn via a sampling cannula (0.7 mm o.d.) implanted chronically in the structure studied; they are aspirated by the high vacuum (10 -9 Torr) of the mass spectrometer and inserted in the analysis chamber where the molecules are ionized, accelerated and separated according to their molecular weight. The specific geometry and the polyethylene membrane of the gas sampling cannula prevent any depletion from occurring; the response time is 51 s for 0 2 and 45 s for CO 2 at 37 °C. The cannula was calibrated before implantation, in a thermoregulated bath containing saline solution saturated with a known mixture of gases (N2, 02, CO2) so that pO 2 and pCO2 could be expressed quantitatively. Arterial partial pressures of oxygen and carbon dioxide (paO2, paCO2) were also continuously and quantitatively measured by mass spectrometry3S. Gases were directly sampled in the aortic blood by a silastic-sheathed cannula (0.4 mm o.d.) inserted in the artery via a Teflon catheter. The time constant of
the measurement was 8-10 s and the previous calibration of the cannula made its quantification possible. Arterial blood pressure (BP) was measured with a Statham transducer via the remaining lumen of the catheter. Furthermore, samples of arterial blood were withdrawn periodically to measure p~,O> PaCO2, pH and Hb with a blood gas analyser (Radiometer ABL 2). The electrocorticogram (ECoG) was also recorded. All the continuous measurements were displayed simultaneously on a polygraph recorder (Beckman R 612).
Experimental procedure All experiments were performed on 25 male 'Fauves de Bourgogne' rabbits, weighing 2.5-3.0 kg. The animals were studied in accordance with the Guiding Principles in the Care and Use of Animals. The experiments were performed in two phases. In phase 1 the rabbit was anaesthetized with diazepam (Valium, 3 mg/kg) and pentobarbital sodium (Nembutal, 35 mg/kg) through the marginal ear vein in order to implant chronically and stereotaxicallv a thermoprobe and a gas sampling cannula in each caudate nucleus. Probes and cortical screws were fixed to the skull with dental cement and were protected by a fibreglass cover. In the second phase, 15 days later (time needed for any possible inflammation or oedema to be resorbedS), anaesthesia was induced by Althestn (CT 1341, Glaxo), a steroid agent with short-lasting action, used at a 2-fold dilution (6 mg/ml) at a dose of 3.5 mg/kg given intravenously. Anaesthesia was then maintained by a perfusion of 30 mg/kg/h during the surgical procedure. The animal was tracheotomized. paralysed (Flaxedil, 5 mg/kg/h i.v.) and artificially ventilated (Braun-50 respirator) with air supplemented with 10% oxygen. The respirator was set to yield paCO2 to about 30 mm Hg and p,~O2 over 150 mm Hg and the ventilation pressure was monitored. Body temperature was kept close to 37 °C by means of a heating pad. A Teflon catheter ( 1.2 mm i.d. ) was introduced into a femoral artery and pushed into the abdominal section of the aorta. It served as a guide for the flexible mass spectrometer cannuta and was used for arterial pressure recording and for arterial blood sampling. The animal was heparinized with
307 400 IU/kg from a 10 mg/ml solution of lyophylized heparin (Choay). The tissues around the incision were infiltrated with a local anaesthetic (2% lidocaine) and protected by cotton wool soaked in physiological solution to prevent pain and desiccation. AIthesin perfusion was then stopped and the animal was left undisturbed. The following variables were continuously recorded: E C o G , BP, paO2, paCO2 and blood flow, pO2, pCO 2 in the caudate nucleus. Arterial blood gases were checked regularly. At the end of the dissipation period of Althesin anaesthesia, i.e. about 30 min after ceasing the perfusion, the stress reaction occurred either spontaneously or was induced by light abdominal tactile stimulations. It was evidenced by both an acute hypertension and an E C o G arousal. The stimulus was maintained during 1 rain, at which time either the stress reaction stopped spontaneously or a bolus of Althesin (2.5 mg i.v.) was injected to stop it. After another period of about 30 min (time necessary for the dissipation of the Althesin), the stress reaction recurred spontaneously or was elicited as previously described. This protocol could be performed throughout the experiment so that not only several control stress reactions could be obtained in the same animal, but also both the intrinsic cerebrovascular effects of various receptor blockers and their effects on the stress reaction could be studied. The following blockers were used: (1) A /~-receptor blocker: propranolol (Avlocardyl, ICI), at a dose of 1 mg/kg; the injection was followed by an equilibration period of 10 min before inducing a stress reaction. Both the dose and the delay were chosen because under these conditions, propranolol has been shown to eliminate the cerebral vasodilatation caused by a/%receptor agonist (isoproterenol, 2.5/~g/kg) in the caudate nucleus 1. (2) An a-receptor blocker: phentolamine (Regitine, Ciba) at a dose of 1 mg/kg and with an equilibration period of 5 rain. Once again, these conditions correspond to those in which the cerebrovascular and the systemic effects of an a-receptor agonist (norepinephrine 0.4/~g/kg) were totally blocked3L (3) A cholinergic receptor blocker: atropine sulfate (Meram) was injected at a dose of 1.5 mg/kg with an equilibration period of 5 rain. Under these conditions, atropine did suppress most of the increase in
CBF induced by a cholinomimetic agent (carbachol, 2.5/,tg/kg/min, i.c.) as shown by the experiments of Aubineau et al. 2. Histology. At the end of each experiment, an intravenous perfusion of Evans blue mixed with venous blood was performed during 20 rain in order to determine if any lesion of the b l o o d - b r a i n barrier (BBB) occurred during the experiments. The animal was then killed with pentobarbital; the whole brain was removed and frozen in chilled isopentane at - 5 0 °C. The brain was cut in sections 40/~m thick in a cryostat (LKB) at - 2 0 °C to verify the location of the probes in the caudate nucleus and to check the integrity of the BBB by fluorescence microscopy of Evans blue around the probe scar. Statistical analysis. Results are given as means + S.E.M. Statistical comparisons were performed with Student's t-test. RESULTS The results reported below were obtained under stable and reproducible physiological conditions as shown by the mean values of the variables measured in 25 rabbits: BP = 90.1 _+ 2.0 mm Hg, paO 2 = 169.3 + 9.2 mm Hg, paCO 2 = 30.1 + 0.7 mm Hg, pH
1so
BP
loo s
PaO 2 35
PaC02
mmHg
3O
],5
2O
PO x
],5
PCO 2
4S *4O
CBF
t
Stress
-~o
t 1rain
Fig. 1. Representative recording showing the effects of immobilization stress in rabbit on local cerebral blood flow and associated variables. ECoG, electrocorticogram; BP, systemic arterial blood pressure; paCO2 and p~O2, partial pressures of CO 2 and 02 in the aorta; pCO 2 and pO2, partial pressures of CO z and O2 in the caudate nucleus; CBF, cerebral blood flow in the caudate nucleus. CBF and BP increased immediately and in parallel at the onset of immobilization stress, pO 2 increased but more slowly than CBF. Variations in CBF are expressed as a percentage of the mean baseline value.
31)8 = 7.40 + 0.01, pO2 = 18.3 + 1.5 mm Hg and p C O 2 = 51.5 _+ 2.2 mm Hg. The cerebrovascular reactivity of the animal was periodically tested during each experiment by administering a gas mixture containing air + 5% CO, for 3 rain.
ECoG
.....................................
] o.smv
150 100 J50
BP
Ps 0 2
PaCO2
Time course of the stress reaction A typical cerebrovascular response to immobilization stress is shown in Fig. 1. As soon as the E C o G was desynchronized, immediate and parallel increases in BP and CBF occurred. A n increase in pO 2 was also noted as a slight increase in paCO2 . The other variables did not vary significantly. Statistical results are given in Fig. 2. Highly significant changes were observed for CBF (+32.8 + 2.1%), BP (+53.1 + 3.4%), pO 2 (+10.8 + 1.7%) and paCO2 (+3.9 +
0.9%). Althesin injected (2.5 rag, i.v.) to stop the stress
P02
PCO2
t'!2
CBF
t Anglot e~sin • i-v 1. S ~lg
i
• I rain
Fig. 3. Representative experiment performed in a rabbit, illustrating the effects of angiotensin II administered intravenously (0,5/~g/kg) on cerebral blood flow. arterial blood pressure and
reaction induced a rapid return of the variables to their baseline values. However, CBF, although lowered by althesin, only regained its basal level 10-20 min after the injection.
associated variables. Same abbreviations as in Fig. 1. Note that angiotensin induced the same increase in pressure as immobilization stress, but not with the same time course and not with the associate increase in flow.
Stability of the stress reaction
onstrated by eliciting stress reaction every 30 rain for 8 h in 3 undrugged rabbits and recording no deviation in their response. Furthermore. if the reaction did not stop spontaneously and if no injection of althesin was performed to arrest the reaction, the increases in CBF, BP and pO2 persisted. The longest duration we allowed to last was 7 min.
Repeated immobilization stress reactions throughout the experiment gave rise to reproducible cerebrovascular reactions with no significant modifications and did not alter baseline levels. This was dem,
% n: 25
S ql 60
8
40
20
S
ns
r~ l
ns
PO z
PC02
0 B P
level
Pa02
PaCO z
CBF
of s i g n i f i c a n c e : S • S = p ~ 0 . 0 0 t ;
ns: non s i g n i f i c a n t
Fig. 2. Statistical summary of the changes induced by immobilization stress in 25 rabbits with respect to arterial blood pressure (BP), partial pressures of 02 and CO 2 in the aorta (p~O2, PaCO2), partial pressures of 02 and CO 2 in the caudate nucleus (pO2, pCOz) and blood flow in the caudate nucleus (CBF). The changes are expressed as a percentage of the mean baseline value. Vertical bars indicate standard errors of the mean (S.E.M.).
Involvement of BP In view of the parallel increases in BP and CBF during immobilization stress, it was hypothesized that BP could be responsible for the cerebral vasodilatation, and that the autoregulatory response did not occur in these conditions. We therefore investigated the influence of the hypertension on the cerebrovascular response. We calculated the correlation coefficient (r) and the least-squares regression line between the increases in CBF and BP (expressed in per cent of their control values) elicited by the stress reaction. The correlation coefficient was 0.122. which indicates that CBF and BP were not correlated. We also compared in each animal the cerebrovascular reaction to stress with that evoked by an intravenous injection of angiotensin I1 (0.5-1.0 ug/kg)
309 giving rise to a quantitatively similar increase in BP (Fig. 3). The time course of the increase in BP was much more sluggish than in case of the stress reaction, and CBF was differently modified by the angiotensin I1 injection (i.e. a biphasic reaction). The ECoG was not modified by this drug.
increase in BP was not significantly different from the one observed in stressful conditions before propranolol injection, the increase in CBF was significantly (P < 0.02) reduced from 36.0 + 4.0% to 20.5 _+ 5.2%, the increase in pO 2 was transformed into a decrease o f - 7 . 0 + 5.8% with a significant difference (P < 0.01) with respect to the change during a control stress, pCO 2 and paO2 were not significantly modified, paCO 2 was increased (2.9 _+ 1.4%) with a nonsignificant difference with regard to the increase observed before propranolol injection.
Involvement oft%receptors In 6 rabbits, intravenous injection of propranolol (1 mg/kg) 10 rain before inducing immobilization stress was without apparent effect on the baseline values, but had the following consequences (statistically summarized in Fig. 4a) on the variations of cerebrovascular variables observed during the stress reaction. The activation of the ECoG was similar, the
a
[~ before propranolol
Involvement of a-receptors In 12 rabbits, intravenous injection of phentolamine (1 mg/kg) 5 rain before inducing immobilization
Flbefore phentolamine
b
• after propranolol ~0
~0 ] 600
n=6 6 0 -s] 1 " n
n
'°20
•
ns
'°1111 20
"
Barfer phentolamino n=12 • * ns
ns
AI
:
•
o BP
PaO 2 PaCO 2 CBF
P02
PC02
BP
PaO 2 PaCO 2 CBF
PO 2
ns
PCO 2
c d
I-Ibefore atropine ~n
I-]before propranoiol+atropine
• after atropine
%
•after propranolol÷atropine
/u
n=7 60 1
n=lO
ns
60 40
•
BP
PaO 2 PaCO 2
CBF
PO 2
PCO 2
n
BP
~
Pa02
~
,
.
•
PaCO 2 CBF
P02
PC02
Level of significance of the differences between the two groups: • • •= p(O.O01; • •:p(O.01 ; e= p(O.02; ns:non sig~flcant Fig. 4. Statistical s u m m a r y of the changes induced by immobilization stress in rabbits, before and after administration of receptor blockers, with respect to the same variables as expressed in Fig. 2. The changes are expressed as a percentage of the m e a n baseline value. Vertical bars indicate standard errors of the mean. a: effects of i.v. injection of a fl-receptor blocker, propranolol ( 1 mg/kg), in 6 rabbits on the variations induced by immobilization stress, b: effects of i.v. injection of an a-receptor blocker, phentolamine (1 mg/kg), in 12 rabbits on the variations induced by immobilization stress, c: effects of i.v. injection of a cholinergic receptor blocker, atropine (1.5 mg/kg), in 7 rabbits on the variations induced by immobilization stress, d: effects of intravenous injection of propranoh)l and phentolamine (same doses as in a and c) in 10 rabbits on the variations induced by immobilization stress.
310 stress led to a slight decrease in BP without any change in other variables+ and provoked modifications (Fig. 4b) of the cerebrovascular response to stress. While the desynchronization of ECoG and the relative increase in BP were unchanged, the cerebral vasodilatation was significantly enhanced (P < 0.01) from 34.9 _+ 2.7% to 51.1 _+ 3.9%. Furthermore, pO 2 was modified and instead of the increase of 12.2 _+ 2.2% observed during the stress reaction before phentolamine injection, a decrease in pO 2 o f - 4 . 1 + 2.8% took place after phentolamine; this difference of pO 2 variation was significant (P < 0.001). Other variables were not significantly modified as during a control stress.
ECo.G.
*J"'J!'[.::".7-'-:[L.':-: ::: :' : ~:;.:",: '7"-:'
150
BP
m,,=mmm,mm~~
In 7 rabbits, intravenous injection of atropine (1.5 mg/kg) 5 min before inducing immobilization stress had no effect on the variables recorded in control conditions but had consequences for the reaction to stress similar to propranolol injection (Fig. 4c). The ECoG activation and acute hypertension were unchanged. The increase in CBF was significantly diminished with regard to the control stress reaction (P < 0.001) from 31.4 + 4.1% to 14.3 + 3.4%. A d e crease in pO 2 o f - 9 . 4 _+ 3.2 was noted which differed significantly (P < 0.01) from the increase of 10.8 _+ 3.6 initially observed. Other variables were not significantly affected.
Involvement of cholinergic + t~-receptors If we combined the blocking effects of propranolol and atropine, injecting them with a 5-min interval, we observed a nearly total abolition of the cerebral vasodilatation during immobilization stress. This can be seen in Fig. 4. The stress induced the same modifications of ECoG and BP, while CBF increased by only 7.3 + 2.2%, which was significantly different (P < 0.001) from the increase of 34.9 + 3.6% noted before the injection of blockers and also from the cerebral vasodilatation of 20.5 + 5.2 and 14.3 + 3.4 induced by immobilization stress after propranoiol and atropine injection respectively. Similarly, pO 2 variations were significantly different (P < 0.001) from those noted in previous conditions. It was decreased by -17.4 + 4.1% whereas in control conditions it was increased by 14.2 + 3.4%. No significant modifications of other variables were observed.
loo 50
~
170
PaO 2
1so 130
PeN
]~o
mm Hg
t"
~
PO2
20 15
co, CBF
1 +5 50 . . . .
o ~ Adrenaline i - v 1.5 pg
Involvement of cholinergic receptors
]o.smv
-4e *
a lmln
Fig. 5. Representative experiment performed in a rabbit, illustrating the effects of adrenaline administered intravenously (0.5 ~g/kg) on cerebral blood flow and associated variables~ Same abbreviations as in Fig. 1. Note that adrenaline induced a decrease in flow and in pO 2 and a slight increase in pCO:.
Involvement of circulating adrenaline In each rabbit, at least two injections of adrenaline (0.3 pg/kg) were performed, one before inducing the stress reaction, the other after. As we previously described3], 35. adrenaline injected intravenously led to a caudate vasoconstriction, an acute hypertension, a decrease in pO 2, and an increase in pCO 2 without any modification of paO2 and paCO:. After the stress reaction, adrenaline induced similar variations of the cerebrovascular variables (Fig. 5) and no significant differences were observed between the two injections.
Histology In each of the rabbits studied, the probes were well located in the caudate nucleus. Fluorescence microscopy revealed that the integrity of the BBB was preserved in the whole caudate nucleus, even in close proximity (about 100/~m) of the probes. DISCUSSION
The present study provides experimental evidence that adrenergic and cholinergic receptors as welt as local metabolism are involved in the cerebral vasodilatation elicited by immobilization stress in rabbits. It also demonstrates that neither circulating adrenaline
311 nor acute hypertension can be directly responsible for this dilatation of brain vessels. Before discussing these results, we should consider the validity of the experimental model and the techniques used. The physiological state of the animals was attested to by the values of the systemic variables which were in the range of those generally described in conscious rabbits 3°. The stress reaction was evidenced by concomitant ECoG activation and BP increase. Special care was taken concerning the wounds to avoid pain, and sufficient time was allowed for dissipation of Althesin anaesthesia. As far as the techniques are concerned, the increase in blood flow measured in the caudate nucleus with thermoclearance during immobilization stress is entirely comparable with the vasodilatation measured in the same structure during an identical experimental procedure with the [HC]ethanol tissue sampling technique 19. Previous results, for example in the case of sympathetic stimulation 36, have already provided evidence that a good correlation exists between the variations measured with the two techniques. Quantitative values of pO 2 in the caudate nucleus measured by mass spectrometry are in the range of those found by polarography 21, suggesting that no major criticism can be raised against this technique. Moreover, the validity of this technique had been previously demonstrated 39 and the quantitative results for pO 2 and pCO~ obtained in the present work closely approximate those described before. The histological study revealed that except for a glial scar (about 100/~m) surrounding the probe, the cerebral tissue was intact and the BBB was not permeable to Evans blue. It should be noted that the gliosis did not affect the measurement because of the special membrane and geometry of the probe, as previously demonstrated 39. Compared to other techniques, advantages of the methods used in this study are the following: they allow to study continuously and simultaneously local cerebral blood flow, pO:, pCO: and arterial pO:, pCO 2 in addition to common systemic variables. This makes it possible to follow the time course of a reaction, to determine how the different variables interact, to repeat the same stimulation in the same animal under different experimental conditions several times (for example, before and after administration of drugs) and also to study a short-lasting phenomenon which is of particular in-
terest in the case of research of the mechanisms involved in the cerebrovascular reaction to immobilization stress. This physiological phenomenon can be compared with the effects of anxiety in human beings 16,41, especially since our results show changes of the same order of magnitude as in the experiments analyzed by these researchers. The continuous recording of P~lO2 and p~CO 2 showed no significant modification during the stress reaction either in undrugged animals or after administration of a receptor blocker. Therefore, it seems unlikely that in this model paCO~ could be responsible for the cerebral vasodilatation observed as suggested by Ohata et al. 27. Furthermore, the time course of the cerebrovascular reaction to stress bore little resemblance to the response to hypercapnia. Regarding the hypertension induced (which never exceeded 160 mm Hg), the possibility that the increase in BP induced damage to the cerebral vasculature with consequent stimulation of cerebral metabolism by, for example, circulating catecholamines, can be rejected in the case of our experimental model, since intravenous adrenaline caused a cerebral vasocontriction before and after the stress reaction: if the BBB had been ruptured by the hypertension induced by stress, adrenaline should have increased cerebral blood flow after the stress reaction. Moreover, fluorescence microscopy did not reveal any opening of the BBB. Furthermore, no increase in the local uptake of [3H]adrenaline was shown in rabbits in identical experimental conditions as in this study ~s. Results obtained by Belova and Jonsson 3 using trypan blue demonstrated that no increase in dye penetration appears in the parenchyma of caudate nucleus during immobilization stress in rats not paralyzed and not previously anaesthetized. Another hypothesis could explain the cerebral vasodilatation during immobilization stress: it would be an activation of the autonomic nervous system responsible for the simultaneous systemic hypertension and the increase in cerebral blood flow. But the receptor blockers did not modify the increase in BP during stress and also no correlation was found between the variation in BP and the variation in flow in the caudate nucleus during the stress reaction. In confirmation, autoregulation was not abolished in animals which had been subjected to stress, since intravenous injection of angiotensin II in these animals
312 induced hypertension with no increase in CBF. Before discussing the influence of receptor blockers on the cerebrovascular reaction to immobilization stress, we should specify that we chose to study blood flow, pO2, and pCO 2 in the caudate nucleus because the vessels irrigating this structure have been shown to be highly innervated 2~, to be provided with vascular receptors 10 and to be particularly sensitive to sympathetic stimulation36 and circulating amines 35. In view of the time course of the CBF increase which seems scarcely compatible with a metabolic phenomenon13,14,22,23, we hypothesized that the nervous system could be directly responsible for the cerebral vasoditatation induced by immobilization stress, but without excluding metabolic factors in the maintenance of hyperaemia when established. Given that a cholinergic cerebral vasodilatator innervation has been demonstratedg,X2,20,26, that the terminals of both the adrenergic and the cholinergic systems form synaptic-type contacts with the arterial smooth muscle fibres 11,15,24,26 and that various g r o u p s 2,7,32,34 have demonstrated the possibility of a cholinergic dilator mechanism in vivo in dogs, rabbits and rats, we investigated by means of adrenergic and cholinergic receptor blockers the possible involvement of the nervous system in the cerebrovascular reaction to immobilization stress. Our results demonstrate that the fladrenergic and muscarinic receptors play a highly significant role in this reaction, but two problems remain unsolved: where are these receptors situated and how are they stimulated? The receptor blockers readily cross the blood-brain barrier in the concentration used so that they can antagonize receptors both in the vessel wall and in the brain parenchyma. However, we can probably exclude an action of circulating catechotamines (which do not cross this barrier) on the caudate parenchyma thereby inducing an increase in metabolism and thus a vasodilatation, since the fluorescence microscopy observations with Evans blue, in agreement with other w o r k 3,28, s e e m to rule out any opening of the BBB; furthermore, the normal action of intravenous adrenaline was to induce a vasoconstriction in the caudate nucleus. However, although circulating catecholamines have no apparent dilating action at the intimal surface of cerebral vessels, they might have a triggering action, either on brain areas lacking a blood-brain barrier, or on peripheral receptors. The latter hypothesis was
proposed by Carlsson et al. 5 in a different model and by Lacombe et al.lS who adrenalectomized rabbits subjected to the same experimental conditions as those described here. In the present study, propranolol which had no effect on the cerebrovascular variables measured, as previously described 6, partly blocked the cerebral vasodilator response to stress, in contradiction with the total blocking effect obtained by Carlsson et al. 5. Probably no direct comparison can be made between the two models because the latter experiments 5 were performed on rats with much longer stimulation, but the two studies demonstrated the involvement of fl-adrenergic receptors in the stress reaction. The enhancement of the cerebral vasodilator response by phentolamine adds further evidence of the involvement of catecholamines in the cerebrovascular reaction to immobilization stress. For all these reasons, it is likely that adrenergic receptors located outside the vessel wall are concerned in the vasodilatation studied, indicating that adrenergic neuronal pathways may be involved: circulating amines seem to have only a modulatory role. Furthermore, Sercombe et al. 36 using the same model as in this study demonstrated that stimulation of the cervical sympathetic nerve, giving rise to adrenergic innervation on cerebral vessels, induces a cerebral vasoconstriction; this is in agreement with the hypothesis of the involvement of a specific intrinsic neuronal pathway. Regarding a cholinergic mechanism, our results suggest that cholinergic receptors are also partly involved in the cerebrovascular reaction studied. Thus the response may be partly mediated by the parasympathetic cholinergic (cerebral) dilator pathways evidenced by several g r o u p s 9,12,2U,26 o r by central cholinergic pathways, for example an intrinsic system originating or projecting through the fastigial nucleus such as suggested by Reis et al? 3. These authors, on stimulating the fastigial nucleus, obtained an increase in BP and a much more pronounced increase in CBF than the increase in glucose consumption would have predicted. This finding apparently involving a cholinergic pathway, can perhaps be related to the results obtained by Pearce et al, 29 who concluded that a short-latency mechanism involving a cholinergic link may mediate cerebral vasodilatation during spontaneous E E G shifts. However. the phenomenon they studied is not exactly similar since
313 it occurs concomitantly with a decrease in blood pres-
the pO 2 increase during stress was significantly lower
sure.
than during control stress, although the C B F increase
The present study performed on immobilized rabbits strongly suggests that neurogenic mechanisms intervene in the regulation of cerebral blood flow in
was higher than that observed in the absence of the blocker. It seems that, as proposed by Siesj640 in the
physiological conditions which may correspond in hu-
an homeostatic mechanism maintaining oxygen availability, so that hypoxia of the cerebral tissue is
man beings to anxiety or recovery after surgery. However, the results also reveal an increase in local
case of hypoxic hypoxia, the hyperaemia represents
avoided during stress. The absence of significant var-
metabolism in this situation, partly responsible for the vasodilatation observed. If we consider the local
iations in p C O 2 can be explained by the very efficient
variations in pO 2 we can see that during the stress reaction induced without any previous injection of re-
In conclusion, the present study of immobilization stress in rabbits provides evidence of the involvement
ceptor blocker, pO2 increased because of the hyper-
of fl-adrenergic and cholinergic receptors and of local
aemia; however, when the increase in flow was diminished by propanolol or atropine, pO 2 decreased with respect to baseline levels. It seems clear that
metabolism in the cerebral vasodilatation which oc-
during the maximal vasodilatation, the high blood flow brought additional available oxygen which more
buffering of CO 2.
curs. It suggests that neurogenic mechanisms, probably intrinsic to brain, intervene in the stress reaction inducing an hyperaemia thus preventing energy failure in brain tissue.
than compensated the increase in metabolism, but the much weaker increase in blood flow after receptor blockade could not ensure the oxygen needs of
ACKNOWLEDGEMENTS
the tissue without a fall in pO2, i.e., more oxygen was consumed than blood flow could provide. O n the oth-
Thanks are due to Mr. J e a n - L o u p Correze for technical assistance, to Mrs. J e a n n e Leizerovici for
er hand, when a-adrenergic receptors were blocked,
typing the manuscript and to Dr. Richard Sercombe for criticism of the manuscript.
REFERENCES 1 Aubineau, P. F., Seylaz, J., Sercombe, R. and Mamo, H., Evidence for regional differences in the effect of beta-adrenergic stimulation on cerebral blood flow, Brain Research, 61 (1973) 153-161. 2 Aubineau, P. F., Sercombe, R. and Seylaz, J., Cholinergic influence on local cerebral blood flow in the rabbit by a direct vasodilator action and an inhibition of the sympatheticmediated vasoconstriction, Brit. J. Pharrnacol., 68 (1980) 449-459. 3 Belova, T. I. and Jonsson, G., Blood-brain barrier permeability and immobilization stress, Acta physiol, scand., 116 (1982) 21-29, 4 Carlsson, C., Hfigerdal, M. and Siesj6, B. K., Increase in cerebral oxygen uptake and blood flow in immobilization stress, Actaphysiol. scand., 95 (1975) 206-208. 5 Carlsson, C., Hfigerdal, M., Kaasik, A. E. and Siesj6, B. K., A catecholamine-mediated increase in cerebral oxygen uptake during immobilization stress in rats, Brain Research, 199 (1977) 223-231. 6 Dahlgren, N., Ingvar, M. and Siesj0, B. K., Effect of propranolol on local cerebral blood flow under normocapnic and hypercapnic conditions, J. Cereb. Blood Flow Metabol., 1 (1981) 429-436. 7 D'Alecy, L. G. and Rose, C. J., Parasympathetic cholinergic control of cerebral blood flow in dogs, Circulat. Res., 41 (1977) 324-331.
8 Edvinsson, L., Nielsen, K. C., Owman, Ch. and West, K. A., Alterations in intracranial pressure, blood brain barrier and brain edema after subchronic implantation of a canula into the brain of conscious animals, A cta physiol, scan&, 82 (1971) 527-531. 9 Edvinsson, L., Nielsen, K. C., Owman, Ch. and Sporrong, B., Cholinergic mechanisms in pial vessels. Histochemistry, electron microscopy and pharmacology, Z. Zellforsch., 134 (1972) 311-325. 10 Edvinsson, L. and Owman, Ch., Pharmacological characterization of adrenergic alpha and beta receptors mediating vasomotor response of cerebral arteries in vitro, Circulat. Res., 35 (1974) 835-849. 11 Edvinsson, L., Falck, B. and Owman, Ch., Possibilities for cholinergic action on smooth musculature and on sympathetic axons in brain vessels mediated by muscarinic and nicotinic receptors, J. Pharmacol. exp. Ther.. 200 (1977) 117-126. 12 Florence, V. M. and Bevan, J. A., Biochemical determination of cholinergic innervation in cerebral arteries, Circulat. Res., 45 (1979) 217-218. 13 Ingvar, D. H. and S6derberg, U., A new method for measuring cerebral blood flow in relation to the electroencephalogram, Electroenceph. clin. Neurophysiol., 8 (1956) 403-412. 14 Ingvar, D. H. and S6derberg, U., Cortical blood flow related to EEG patterns evoked by stimulation of brain stem, Acta physiol, scand., 42 (1958) 130-143.
314 15 Iwayama, T., Furness, J. B. and Burnstock, G., Dual adrenergic and cholinergic innervation of the cerebral arteries of the rat, Circular. Res., 26 (1970) 635-646. 16 Kety, S. S., Circulation and metabolism of the human brain in health and disease, Amer. J. Med., 8 (1950) 205-217. 17 King, B. D., Sokoloff, L. and Wechsler, R. L., The effects of t.-epinephrine and L-norepinephrine upon cerebral circulation and metabolism in man, J. clin. Invest., 31 11952) 273-279. 18 Lacombe, P., Reynier-Rebuffel. A. M. and Seylaz, J., Cerebral uptake of circulating catecholamines during stressinduced acute hypertension. In D. D. Heistad and M. L. Marcus (Eds.), Cerebral Blood Flow: Effects of Nerves and Neurotransmitters. Elsevier, New York. 1982, pp. 343-348. 19 Lacombe, P. and Seylaz, J., Significance of the cerebrovascular effects of immobilization stress in the rabbit, J. Cereb. Blood Flow Metabol., in press. 20 Lavrentieva. N. B., Mchedlishvili, G. I. and Plechkova, E. K., Distribution and activity of cholinesterase in the nervous structures of the pial arteries (a histochemical study), ByulL eksp. Biol. Med., 64 (1968) 110-113. 21 Leniger-Follert, E., Wrabetz, W. and Liibbers, D. W., Local tissue pO 2 and microflow of the brain cortex under varying arterial oxygen pressure. In J. Grote, D. Reneau and G. Thews (Eds.), Oxygen Transport to Tissue, Vol. 75, Plenum Press, New York. 1976, pp. 361-367. 22 Meyer, J. S., Nomura, F., Sakamoto, K. and Kondo, A., Effect of stimulation of the brain stem reticular formation on cerebral blood flow and oxygen consumption, Electroenceph. clin. Neurophysiol., 26 11969) 125-I32. 23 Meyer, J. S., Teraura, T., Sakamoto, K~ and Kondo, A., Central neurogenic control of cerebral blood flow, Neurology. 21 (1971) 247-263. 24 Nelson, E. and Rennels, M., Neuromuscular contacts in intracranial arteries of the cat, Science, 167 (1969) 301-302. 25 Nielsen, K. C. and Owman, Ch., Adrenergic innervation of pial arteries related to the circle of Willis in the cat, Brain Research, 6 (1967) 773-776. 26 Nielsen, K. C., Owman, Ch. and Sporrong, B., Ultrastructure of the autonomic innervation apparatus in the main pial arteries of rats and cats, Brain Research, 27 (1971) 25-32. 27 Ohata, M., Fredericks, W. R., Sundaram, U. and Rapoport, S. I.. Effects of immobilization stress on regional cerebral blood flow in the conscious rat, J. Cereb. Blood Flow Metabol., 1 (1981) 187-194. 28 Ohno, K., Pettigrew, K. D. and Rapoport, S. I., Lower limits of cerebrovascular permeability to non electrolytes in the conscious rat, Amer. J. Physiol., 235 (1978) H299-H307. 29 Pearce, W. J., Scremin, O. U., Sonnenschein, R. R. and Rubinstein, E. H., The electroencephalogram, blood flow and oxygen uptake in rabbit cerebrum, J. Cereb. Blood
Flow Metabol.. 1 ( 1981 ) 419-428. 30 Peperkamp. J. P. C.. Cerebral Blood Flon m the Rabbit. Normal Values and the Effects of Some Anesthetics and Vasoactive Substances on the Central Blood Flow. In: l"hesis in Neurobiology: Collected Reprints. Vol. 10. Centra] Institute for Brain Research. Amsterdam. 1972. pp. 1- 140. 31 Pinard. E. and Sevlaz. J.. Intracerebral gas partial pressures changes under vasoactive drugs: a mass spectrometry study, Pfliigers Arch. ges. Physiol., 375 (1978) 25-3t;. 32 Pinard. E.. Purves. M J.. Seylaz. J. and Vasquez. J.. The cholinergic pathway to cerebral blood vessels. II Physiok)gical studies. Pfli~gers Arch. ees. PhysioL, 379 /1979~ 165-172. 33 Reis. D. J., ladecola. C.. MacKenzie. E. T.. Mori. M.. Nakai. M. and Tucker. L. W.. Primary and metabolically coupled cerebrovascular dilation elicited by stimulation of two intrinsic systems of brain. In D. D. Heistad and M. k Marcus (Eds.), Cerebral Blood Flow: Effects of Nerves and Neurotransmuters Elsevier. New York_ 1982. pp. 475-484. 34 Scremin. O. U.. Rubinstein. E. H. and Sonnenschein. R. R. Cholinerglc modulation of the cerebrovascular response to CO z in the rabbit. In Ch. Owman and L. Edvinsson (Eds.), Neurogenic Control c~f the Brain Circulation Pergamon, New York. 1977. pp. 471-481. 35 Sercombe. R.. Aubineau. P.. Edvinsson. L.. Mamo. H.. Owman. Ch.. Pinard. E. and Seylaz. ,I.. Neurogenic influence on local cerebral blood flow. Neurology, 25 (1975] 954-963. 36 Sercombe. R.. Lacombe. P.. Aubineau, P . Mamo. H.. Pinard. E.. Reynier-Rebuffel. A. M. and Seylaz. J.. Is there an active mechanism limiting the influence of the sympathetic system on the cerebral vascular bed? Evidence for vasomotor escape from sympathetic stimulation in the rabbit, Brain Research, 164 (1979) 81-102. 37 Seylaz, J., Aubineau, P. F., Correze, J. L. and Mamo, H., Techniques for continuous measurement of local cerebral blood flow, PaO2, PaCO2 and blood pressure in the non anesthetized animal, Pfliigers Arch. ges. Physiol.. 340 (1973) 175-180. 38 Seylaz, J., Pinard, E., Correze, J. L., Aubineau, P. F. and Mamo, H., Quantitative continuous measurement of blood gas tensions by mass spectrometry, J. appl. Physiol., 37 (1974) 937-941. 39 Seylaz, J. and Pinard, E., Continuous measurement of gas partial pressures in intracerebrat tissue, J. appl. Physiol., 4411978) 528-533. 40 Siesjo, B. K., Brain Energy Metabolism, Wiley, New York, 1978, 285 and 422 pp. 41 Sokoloff, L., Cerebral circulation and behavior in man: strategy and findings. In A. J. Mandell and M. P. Mandell (Eds.), Psychochemical Research in Man, Academic, New York, 1969, pp. 237-252.
3(15
Elsevier BRE I0903
Intrinsic and Extrinsic Mechanisms Involved in the Cerebrovascular Reaction Elicited by Immobilization Stress in Rabbits E. PINARD, P. LACOMBE, A. M. REYNIER-REBUFFEL and J. SEYLAZ Laboratoire de Physiologie et Physiopathologie COrObrovasculaire, E. R.A. 361 C. N. R. S.. U. 182 l. N. S. E. R.M., Universit~ Paris VII, Paris (France
(Accepted November 6th, 1984) KCv words: cerebral blood flow - - cerebral metabolism - - pO 2- - pCO z - - immobilization stress - - neurogenic control
Variations in cerebral blood flow and partial pressures of oxygen and carbon dioxide (pO2, pCO2) were studied in rabbits during short-duration (1 min) immobilization stress. The techniques used enabled us to determine these variables locally in the caudate nucleus in a continuous, simultaneous and quantitative fashion. It could be shown that cerebral blood flow and arterial blood pressure increased in parallel immediately after inducing the stress reaction, and that pO 2 increased further, indicating that cerebral oxygen supply is maintained by the hyperaemia. Previous administration of a fl-receptor blocker or of a cholinergic receptor blocker significantly diminished the cerebrovascular reaction to stress, inducing a decrease in pO 2 during the reaction. Administration of both blockers nearly abolished the cerebral vasodilatation studied. Previous administration of an a-receptor blocker enhanced the reactive hyperaemia. No disturbance of the blood-brain barrier could be observed in rabbits subjected to stress. Intravenous injection of adrenaline, as well as angiotensin II inducing similar increases in blood pressure, had no comparable effect on the blood flow. The conclusion is that in this model of anxiety, neurogenic mechanisms are involved in the provision of a sufficient oxygen supply to the brain.
INTRODUCTION
stress reaction were still unknown since they had not been able to prove that catecholamines act on specif-
Cerebral circulation and metabolism are not only a
ic brain areas or on peripheral receptors that second-
subject of interest with regard to cerebrovascular dis-
arily influence neuronal activity. Contradictory re-
eases, but also in relation to critical physiological sit-
sults were obtained by O h at a et al. 27 working on re-
uations, In fact, more than 30 years ago, Kety 16 dem-
gional cerebral blood flow in the spontaneously
onstrated that anxiety in human beings can be asso-
breathing rat: they d e m o n s t r a t e d that the p~CO2 is
ciated with an increase in cerebral metabolic rate.
responsible for at most 20% of the variation of cere-
F u r t h e r m o r e , an increase in cerebral blood flow and
bral blood flow during immobilization stress. In view
oxygen consumption was observed during infusion of
of this controversy, the purpose of this study was to
epinephrine in a dose which raised arterial pressure
investigate the possible i n v o l v e m e n t of intrinsic and
and caused symptoms of anxiety17. M o r e recently,
extrinsic mechanisms in the variations of local cere-
Carlsson et al.4 d e v e l o p e d an experimental model of
bral circulation and metabolism during immobiliza-
immobilization stress in rats which provided evidence
tion stress. Accordingly we made continuous meas-
that stressful situations may cause significant increas-
urements on the time course of the p h e n o m e n o n and
es in global cerebral blood flow and metabolism. This
on the possible interference of various receptor
model enabled these workers to study the mechanisms of these increases and to d e t e r m i n e that they
blockers with the stress reaction. Several systemic and cerebrovascular variables were m o n i t o r e d simul-
were mediated via release of catecholamines from
taneously throughout experiments in which short-
the adrenal glands 5. H o w e v e r , they concluded that
lasting (1 min) immobilization stress reactions were
the
elicited periodically.
detailed
mechanisms
of the
cerebrovascular
Correspondence: E. Pinard, 10, avenue de Verdun, 75010 Paris, France.
0006-8993/85/$113.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
306 MATERIALS AND METHODS
Techniques Cerebral blood flow (CBF) was measured continuously in the caudate nucleus by means of a thermoclearance technique 37. This method is based on the thermal conductivity of the tissue which varies when blood flow varies. It requires the use of two heat-sensitive probes, one being heated to about one degree above the temperature of the second, unheated probe, used as a reference. A feed-back system maintains the temperature difference constant and the heating current maintaining this temperature difference is linearly related to the variations in blood flow. These variations can be expressed as per cent deviations about the baseline flow since at the end of the experiment, when the animal was killed with an overdose of i.v. pentobarbital, the zero level of cerebral blood flow was obtained. Response time of the blood flow measurement was 1-2 s. The thermoprobe consisted of a glass tube (0.7 m m o.d.) containing a thermistor, around which a heating coil was wound. Tissue partial pressures of oxygen and carbon dioxide (pO 2, pCO2) were quantitatively and continuously measured by mass spectrometry in the same nucleus. A detailed analysis of the theory and description of the technique used have already been published 39. Briefly, gases are withdrawn via a sampling cannula (0.7 mm o.d.) implanted chronically in the structure studied; they are aspirated by the high vacuum (10 -9 Torr) of the mass spectrometer and inserted in the analysis chamber where the molecules are ionized, accelerated and separated according to their molecular weight. The specific geometry and the polyethylene membrane of the gas sampling cannula prevent any depletion from occurring; the response time is 51 s for 0 2 and 45 s for CO 2 at 37 °C. The cannula was calibrated before implantation, in a thermoregulated bath containing saline solution saturated with a known mixture of gases (N2, 02, CO2) so that pO 2 and pCO2 could be expressed quantitatively. Arterial partial pressures of oxygen and carbon dioxide (paO2, paCO2) were also continuously and quantitatively measured by mass spectrometry3S. Gases were directly sampled in the aortic blood by a silastic-sheathed cannula (0.4 mm o.d.) inserted in the artery via a Teflon catheter. The time constant of
the measurement was 8-10 s and the previous calibration of the cannula made its quantification possible. Arterial blood pressure (BP) was measured with a Statham transducer via the remaining lumen of the catheter. Furthermore, samples of arterial blood were withdrawn periodically to measure p~,O> PaCO2, pH and Hb with a blood gas analyser (Radiometer ABL 2). The electrocorticogram (ECoG) was also recorded. All the continuous measurements were displayed simultaneously on a polygraph recorder (Beckman R 612).
Experimental procedure All experiments were performed on 25 male 'Fauves de Bourgogne' rabbits, weighing 2.5-3.0 kg. The animals were studied in accordance with the Guiding Principles in the Care and Use of Animals. The experiments were performed in two phases. In phase 1 the rabbit was anaesthetized with diazepam (Valium, 3 mg/kg) and pentobarbital sodium (Nembutal, 35 mg/kg) through the marginal ear vein in order to implant chronically and stereotaxicallv a thermoprobe and a gas sampling cannula in each caudate nucleus. Probes and cortical screws were fixed to the skull with dental cement and were protected by a fibreglass cover. In the second phase, 15 days later (time needed for any possible inflammation or oedema to be resorbedS), anaesthesia was induced by Althestn (CT 1341, Glaxo), a steroid agent with short-lasting action, used at a 2-fold dilution (6 mg/ml) at a dose of 3.5 mg/kg given intravenously. Anaesthesia was then maintained by a perfusion of 30 mg/kg/h during the surgical procedure. The animal was tracheotomized. paralysed (Flaxedil, 5 mg/kg/h i.v.) and artificially ventilated (Braun-50 respirator) with air supplemented with 10% oxygen. The respirator was set to yield paCO2 to about 30 mm Hg and p,~O2 over 150 mm Hg and the ventilation pressure was monitored. Body temperature was kept close to 37 °C by means of a heating pad. A Teflon catheter ( 1.2 mm i.d. ) was introduced into a femoral artery and pushed into the abdominal section of the aorta. It served as a guide for the flexible mass spectrometer cannuta and was used for arterial pressure recording and for arterial blood sampling. The animal was heparinized with
307 400 IU/kg from a 10 mg/ml solution of lyophylized heparin (Choay). The tissues around the incision were infiltrated with a local anaesthetic (2% lidocaine) and protected by cotton wool soaked in physiological solution to prevent pain and desiccation. AIthesin perfusion was then stopped and the animal was left undisturbed. The following variables were continuously recorded: E C o G , BP, paO2, paCO2 and blood flow, pO2, pCO 2 in the caudate nucleus. Arterial blood gases were checked regularly. At the end of the dissipation period of Althesin anaesthesia, i.e. about 30 min after ceasing the perfusion, the stress reaction occurred either spontaneously or was induced by light abdominal tactile stimulations. It was evidenced by both an acute hypertension and an E C o G arousal. The stimulus was maintained during 1 rain, at which time either the stress reaction stopped spontaneously or a bolus of Althesin (2.5 mg i.v.) was injected to stop it. After another period of about 30 min (time necessary for the dissipation of the Althesin), the stress reaction recurred spontaneously or was elicited as previously described. This protocol could be performed throughout the experiment so that not only several control stress reactions could be obtained in the same animal, but also both the intrinsic cerebrovascular effects of various receptor blockers and their effects on the stress reaction could be studied. The following blockers were used: (1) A /~-receptor blocker: propranolol (Avlocardyl, ICI), at a dose of 1 mg/kg; the injection was followed by an equilibration period of 10 min before inducing a stress reaction. Both the dose and the delay were chosen because under these conditions, propranolol has been shown to eliminate the cerebral vasodilatation caused by a/%receptor agonist (isoproterenol, 2.5/~g/kg) in the caudate nucleus 1. (2) An a-receptor blocker: phentolamine (Regitine, Ciba) at a dose of 1 mg/kg and with an equilibration period of 5 rain. Once again, these conditions correspond to those in which the cerebrovascular and the systemic effects of an a-receptor agonist (norepinephrine 0.4/~g/kg) were totally blocked3L (3) A cholinergic receptor blocker: atropine sulfate (Meram) was injected at a dose of 1.5 mg/kg with an equilibration period of 5 rain. Under these conditions, atropine did suppress most of the increase in
CBF induced by a cholinomimetic agent (carbachol, 2.5/,tg/kg/min, i.c.) as shown by the experiments of Aubineau et al. 2. Histology. At the end of each experiment, an intravenous perfusion of Evans blue mixed with venous blood was performed during 20 rain in order to determine if any lesion of the b l o o d - b r a i n barrier (BBB) occurred during the experiments. The animal was then killed with pentobarbital; the whole brain was removed and frozen in chilled isopentane at - 5 0 °C. The brain was cut in sections 40/~m thick in a cryostat (LKB) at - 2 0 °C to verify the location of the probes in the caudate nucleus and to check the integrity of the BBB by fluorescence microscopy of Evans blue around the probe scar. Statistical analysis. Results are given as means + S.E.M. Statistical comparisons were performed with Student's t-test. RESULTS The results reported below were obtained under stable and reproducible physiological conditions as shown by the mean values of the variables measured in 25 rabbits: BP = 90.1 _+ 2.0 mm Hg, paO 2 = 169.3 + 9.2 mm Hg, paCO 2 = 30.1 + 0.7 mm Hg, pH
1so
BP
loo s
PaO 2 35
PaC02
mmHg
3O
],5
2O
PO x
],5
PCO 2
4S *4O
CBF
t
Stress
-~o
t 1rain
Fig. 1. Representative recording showing the effects of immobilization stress in rabbit on local cerebral blood flow and associated variables. ECoG, electrocorticogram; BP, systemic arterial blood pressure; paCO2 and p~O2, partial pressures of CO 2 and 02 in the aorta; pCO 2 and pO2, partial pressures of CO z and O2 in the caudate nucleus; CBF, cerebral blood flow in the caudate nucleus. CBF and BP increased immediately and in parallel at the onset of immobilization stress, pO 2 increased but more slowly than CBF. Variations in CBF are expressed as a percentage of the mean baseline value.
31)8 = 7.40 + 0.01, pO2 = 18.3 + 1.5 mm Hg and p C O 2 = 51.5 _+ 2.2 mm Hg. The cerebrovascular reactivity of the animal was periodically tested during each experiment by administering a gas mixture containing air + 5% CO, for 3 rain.
ECoG
.....................................
] o.smv
150 100 J50
BP
Ps 0 2
PaCO2
Time course of the stress reaction A typical cerebrovascular response to immobilization stress is shown in Fig. 1. As soon as the E C o G was desynchronized, immediate and parallel increases in BP and CBF occurred. A n increase in pO 2 was also noted as a slight increase in paCO2 . The other variables did not vary significantly. Statistical results are given in Fig. 2. Highly significant changes were observed for CBF (+32.8 + 2.1%), BP (+53.1 + 3.4%), pO 2 (+10.8 + 1.7%) and paCO2 (+3.9 +
0.9%). Althesin injected (2.5 rag, i.v.) to stop the stress
P02
PCO2
t'!2
CBF
t Anglot e~sin • i-v 1. S ~lg
i
• I rain
Fig. 3. Representative experiment performed in a rabbit, illustrating the effects of angiotensin II administered intravenously (0,5/~g/kg) on cerebral blood flow. arterial blood pressure and
reaction induced a rapid return of the variables to their baseline values. However, CBF, although lowered by althesin, only regained its basal level 10-20 min after the injection.
associated variables. Same abbreviations as in Fig. 1. Note that angiotensin induced the same increase in pressure as immobilization stress, but not with the same time course and not with the associate increase in flow.
Stability of the stress reaction
onstrated by eliciting stress reaction every 30 rain for 8 h in 3 undrugged rabbits and recording no deviation in their response. Furthermore. if the reaction did not stop spontaneously and if no injection of althesin was performed to arrest the reaction, the increases in CBF, BP and pO2 persisted. The longest duration we allowed to last was 7 min.
Repeated immobilization stress reactions throughout the experiment gave rise to reproducible cerebrovascular reactions with no significant modifications and did not alter baseline levels. This was dem,
% n: 25
S ql 60
8
40
20
S
ns
r~ l
ns
PO z
PC02
0 B P
level
Pa02
PaCO z
CBF
of s i g n i f i c a n c e : S • S = p ~ 0 . 0 0 t ;
ns: non s i g n i f i c a n t
Fig. 2. Statistical summary of the changes induced by immobilization stress in 25 rabbits with respect to arterial blood pressure (BP), partial pressures of 02 and CO 2 in the aorta (p~O2, PaCO2), partial pressures of 02 and CO 2 in the caudate nucleus (pO2, pCOz) and blood flow in the caudate nucleus (CBF). The changes are expressed as a percentage of the mean baseline value. Vertical bars indicate standard errors of the mean (S.E.M.).
Involvement of BP In view of the parallel increases in BP and CBF during immobilization stress, it was hypothesized that BP could be responsible for the cerebral vasodilatation, and that the autoregulatory response did not occur in these conditions. We therefore investigated the influence of the hypertension on the cerebrovascular response. We calculated the correlation coefficient (r) and the least-squares regression line between the increases in CBF and BP (expressed in per cent of their control values) elicited by the stress reaction. The correlation coefficient was 0.122. which indicates that CBF and BP were not correlated. We also compared in each animal the cerebrovascular reaction to stress with that evoked by an intravenous injection of angiotensin I1 (0.5-1.0 ug/kg)
309 giving rise to a quantitatively similar increase in BP (Fig. 3). The time course of the increase in BP was much more sluggish than in case of the stress reaction, and CBF was differently modified by the angiotensin I1 injection (i.e. a biphasic reaction). The ECoG was not modified by this drug.
increase in BP was not significantly different from the one observed in stressful conditions before propranolol injection, the increase in CBF was significantly (P < 0.02) reduced from 36.0 + 4.0% to 20.5 _+ 5.2%, the increase in pO 2 was transformed into a decrease o f - 7 . 0 + 5.8% with a significant difference (P < 0.01) with respect to the change during a control stress, pCO 2 and paO2 were not significantly modified, paCO 2 was increased (2.9 _+ 1.4%) with a nonsignificant difference with regard to the increase observed before propranolol injection.
Involvement oft%receptors In 6 rabbits, intravenous injection of propranolol (1 mg/kg) 10 rain before inducing immobilization stress was without apparent effect on the baseline values, but had the following consequences (statistically summarized in Fig. 4a) on the variations of cerebrovascular variables observed during the stress reaction. The activation of the ECoG was similar, the
a
[~ before propranolol
Involvement of a-receptors In 12 rabbits, intravenous injection of phentolamine (1 mg/kg) 5 rain before inducing immobilization
Flbefore phentolamine
b
• after propranolol ~0
~0 ] 600
n=6 6 0 -s] 1 " n
n
'°20
•
ns
'°1111 20
"
Barfer phentolamino n=12 • * ns
ns
AI
:
•
o BP
PaO 2 PaCO 2 CBF
P02
PC02
BP
PaO 2 PaCO 2 CBF
PO 2
ns
PCO 2
c d
I-Ibefore atropine ~n
I-]before propranoiol+atropine
• after atropine
%
•after propranolol÷atropine
/u
n=7 60 1
n=lO
ns
60 40
•
BP
PaO 2 PaCO 2
CBF
PO 2
PCO 2
n
BP
~
Pa02
~
,
.
•
PaCO 2 CBF
P02
PC02
Level of significance of the differences between the two groups: • • •= p(O.O01; • •:p(O.01 ; e= p(O.02; ns:non sig~flcant Fig. 4. Statistical s u m m a r y of the changes induced by immobilization stress in rabbits, before and after administration of receptor blockers, with respect to the same variables as expressed in Fig. 2. The changes are expressed as a percentage of the m e a n baseline value. Vertical bars indicate standard errors of the mean. a: effects of i.v. injection of a fl-receptor blocker, propranolol ( 1 mg/kg), in 6 rabbits on the variations induced by immobilization stress, b: effects of i.v. injection of an a-receptor blocker, phentolamine (1 mg/kg), in 12 rabbits on the variations induced by immobilization stress, c: effects of i.v. injection of a cholinergic receptor blocker, atropine (1.5 mg/kg), in 7 rabbits on the variations induced by immobilization stress, d: effects of intravenous injection of propranoh)l and phentolamine (same doses as in a and c) in 10 rabbits on the variations induced by immobilization stress.
310 stress led to a slight decrease in BP without any change in other variables+ and provoked modifications (Fig. 4b) of the cerebrovascular response to stress. While the desynchronization of ECoG and the relative increase in BP were unchanged, the cerebral vasodilatation was significantly enhanced (P < 0.01) from 34.9 _+ 2.7% to 51.1 _+ 3.9%. Furthermore, pO 2 was modified and instead of the increase of 12.2 _+ 2.2% observed during the stress reaction before phentolamine injection, a decrease in pO 2 o f - 4 . 1 + 2.8% took place after phentolamine; this difference of pO 2 variation was significant (P < 0.001). Other variables were not significantly modified as during a control stress.
ECo.G.
*J"'J!'[.::".7-'-:[L.':-: ::: :' : ~:;.:",: '7"-:'
150
BP
m,,=mmm,mm~~
In 7 rabbits, intravenous injection of atropine (1.5 mg/kg) 5 min before inducing immobilization stress had no effect on the variables recorded in control conditions but had consequences for the reaction to stress similar to propranolol injection (Fig. 4c). The ECoG activation and acute hypertension were unchanged. The increase in CBF was significantly diminished with regard to the control stress reaction (P < 0.001) from 31.4 + 4.1% to 14.3 + 3.4%. A d e crease in pO 2 o f - 9 . 4 _+ 3.2 was noted which differed significantly (P < 0.01) from the increase of 10.8 _+ 3.6 initially observed. Other variables were not significantly affected.
Involvement of cholinergic + t~-receptors If we combined the blocking effects of propranolol and atropine, injecting them with a 5-min interval, we observed a nearly total abolition of the cerebral vasodilatation during immobilization stress. This can be seen in Fig. 4. The stress induced the same modifications of ECoG and BP, while CBF increased by only 7.3 + 2.2%, which was significantly different (P < 0.001) from the increase of 34.9 + 3.6% noted before the injection of blockers and also from the cerebral vasodilatation of 20.5 + 5.2 and 14.3 + 3.4 induced by immobilization stress after propranoiol and atropine injection respectively. Similarly, pO 2 variations were significantly different (P < 0.001) from those noted in previous conditions. It was decreased by -17.4 + 4.1% whereas in control conditions it was increased by 14.2 + 3.4%. No significant modifications of other variables were observed.
loo 50
~
170
PaO 2
1so 130
PeN
]~o
mm Hg
t"
~
PO2
20 15
co, CBF
1 +5 50 . . . .
o ~ Adrenaline i - v 1.5 pg
Involvement of cholinergic receptors
]o.smv
-4e *
a lmln
Fig. 5. Representative experiment performed in a rabbit, illustrating the effects of adrenaline administered intravenously (0.5 ~g/kg) on cerebral blood flow and associated variables~ Same abbreviations as in Fig. 1. Note that adrenaline induced a decrease in flow and in pO 2 and a slight increase in pCO:.
Involvement of circulating adrenaline In each rabbit, at least two injections of adrenaline (0.3 pg/kg) were performed, one before inducing the stress reaction, the other after. As we previously described3], 35. adrenaline injected intravenously led to a caudate vasoconstriction, an acute hypertension, a decrease in pO 2, and an increase in pCO 2 without any modification of paO2 and paCO:. After the stress reaction, adrenaline induced similar variations of the cerebrovascular variables (Fig. 5) and no significant differences were observed between the two injections.
Histology In each of the rabbits studied, the probes were well located in the caudate nucleus. Fluorescence microscopy revealed that the integrity of the BBB was preserved in the whole caudate nucleus, even in close proximity (about 100/~m) of the probes. DISCUSSION
The present study provides experimental evidence that adrenergic and cholinergic receptors as welt as local metabolism are involved in the cerebral vasodilatation elicited by immobilization stress in rabbits. It also demonstrates that neither circulating adrenaline
311 nor acute hypertension can be directly responsible for this dilatation of brain vessels. Before discussing these results, we should consider the validity of the experimental model and the techniques used. The physiological state of the animals was attested to by the values of the systemic variables which were in the range of those generally described in conscious rabbits 3°. The stress reaction was evidenced by concomitant ECoG activation and BP increase. Special care was taken concerning the wounds to avoid pain, and sufficient time was allowed for dissipation of Althesin anaesthesia. As far as the techniques are concerned, the increase in blood flow measured in the caudate nucleus with thermoclearance during immobilization stress is entirely comparable with the vasodilatation measured in the same structure during an identical experimental procedure with the [HC]ethanol tissue sampling technique 19. Previous results, for example in the case of sympathetic stimulation 36, have already provided evidence that a good correlation exists between the variations measured with the two techniques. Quantitative values of pO 2 in the caudate nucleus measured by mass spectrometry are in the range of those found by polarography 21, suggesting that no major criticism can be raised against this technique. Moreover, the validity of this technique had been previously demonstrated 39 and the quantitative results for pO 2 and pCO~ obtained in the present work closely approximate those described before. The histological study revealed that except for a glial scar (about 100/~m) surrounding the probe, the cerebral tissue was intact and the BBB was not permeable to Evans blue. It should be noted that the gliosis did not affect the measurement because of the special membrane and geometry of the probe, as previously demonstrated 39. Compared to other techniques, advantages of the methods used in this study are the following: they allow to study continuously and simultaneously local cerebral blood flow, pO:, pCO: and arterial pO:, pCO 2 in addition to common systemic variables. This makes it possible to follow the time course of a reaction, to determine how the different variables interact, to repeat the same stimulation in the same animal under different experimental conditions several times (for example, before and after administration of drugs) and also to study a short-lasting phenomenon which is of particular in-
terest in the case of research of the mechanisms involved in the cerebrovascular reaction to immobilization stress. This physiological phenomenon can be compared with the effects of anxiety in human beings 16,41, especially since our results show changes of the same order of magnitude as in the experiments analyzed by these researchers. The continuous recording of P~lO2 and p~CO 2 showed no significant modification during the stress reaction either in undrugged animals or after administration of a receptor blocker. Therefore, it seems unlikely that in this model paCO~ could be responsible for the cerebral vasodilatation observed as suggested by Ohata et al. 27. Furthermore, the time course of the cerebrovascular reaction to stress bore little resemblance to the response to hypercapnia. Regarding the hypertension induced (which never exceeded 160 mm Hg), the possibility that the increase in BP induced damage to the cerebral vasculature with consequent stimulation of cerebral metabolism by, for example, circulating catecholamines, can be rejected in the case of our experimental model, since intravenous adrenaline caused a cerebral vasocontriction before and after the stress reaction: if the BBB had been ruptured by the hypertension induced by stress, adrenaline should have increased cerebral blood flow after the stress reaction. Moreover, fluorescence microscopy did not reveal any opening of the BBB. Furthermore, no increase in the local uptake of [3H]adrenaline was shown in rabbits in identical experimental conditions as in this study ~s. Results obtained by Belova and Jonsson 3 using trypan blue demonstrated that no increase in dye penetration appears in the parenchyma of caudate nucleus during immobilization stress in rats not paralyzed and not previously anaesthetized. Another hypothesis could explain the cerebral vasodilatation during immobilization stress: it would be an activation of the autonomic nervous system responsible for the simultaneous systemic hypertension and the increase in cerebral blood flow. But the receptor blockers did not modify the increase in BP during stress and also no correlation was found between the variation in BP and the variation in flow in the caudate nucleus during the stress reaction. In confirmation, autoregulation was not abolished in animals which had been subjected to stress, since intravenous injection of angiotensin II in these animals
312 induced hypertension with no increase in CBF. Before discussing the influence of receptor blockers on the cerebrovascular reaction to immobilization stress, we should specify that we chose to study blood flow, pO2, and pCO 2 in the caudate nucleus because the vessels irrigating this structure have been shown to be highly innervated 2~, to be provided with vascular receptors 10 and to be particularly sensitive to sympathetic stimulation36 and circulating amines 35. In view of the time course of the CBF increase which seems scarcely compatible with a metabolic phenomenon13,14,22,23, we hypothesized that the nervous system could be directly responsible for the cerebral vasoditatation induced by immobilization stress, but without excluding metabolic factors in the maintenance of hyperaemia when established. Given that a cholinergic cerebral vasodilatator innervation has been demonstratedg,X2,20,26, that the terminals of both the adrenergic and the cholinergic systems form synaptic-type contacts with the arterial smooth muscle fibres 11,15,24,26 and that various g r o u p s 2,7,32,34 have demonstrated the possibility of a cholinergic dilator mechanism in vivo in dogs, rabbits and rats, we investigated by means of adrenergic and cholinergic receptor blockers the possible involvement of the nervous system in the cerebrovascular reaction to immobilization stress. Our results demonstrate that the fladrenergic and muscarinic receptors play a highly significant role in this reaction, but two problems remain unsolved: where are these receptors situated and how are they stimulated? The receptor blockers readily cross the blood-brain barrier in the concentration used so that they can antagonize receptors both in the vessel wall and in the brain parenchyma. However, we can probably exclude an action of circulating catechotamines (which do not cross this barrier) on the caudate parenchyma thereby inducing an increase in metabolism and thus a vasodilatation, since the fluorescence microscopy observations with Evans blue, in agreement with other w o r k 3,28, s e e m to rule out any opening of the BBB; furthermore, the normal action of intravenous adrenaline was to induce a vasoconstriction in the caudate nucleus. However, although circulating catecholamines have no apparent dilating action at the intimal surface of cerebral vessels, they might have a triggering action, either on brain areas lacking a blood-brain barrier, or on peripheral receptors. The latter hypothesis was
proposed by Carlsson et al. 5 in a different model and by Lacombe et al.lS who adrenalectomized rabbits subjected to the same experimental conditions as those described here. In the present study, propranolol which had no effect on the cerebrovascular variables measured, as previously described 6, partly blocked the cerebral vasodilator response to stress, in contradiction with the total blocking effect obtained by Carlsson et al. 5. Probably no direct comparison can be made between the two models because the latter experiments 5 were performed on rats with much longer stimulation, but the two studies demonstrated the involvement of fl-adrenergic receptors in the stress reaction. The enhancement of the cerebral vasodilator response by phentolamine adds further evidence of the involvement of catecholamines in the cerebrovascular reaction to immobilization stress. For all these reasons, it is likely that adrenergic receptors located outside the vessel wall are concerned in the vasodilatation studied, indicating that adrenergic neuronal pathways may be involved: circulating amines seem to have only a modulatory role. Furthermore, Sercombe et al. 36 using the same model as in this study demonstrated that stimulation of the cervical sympathetic nerve, giving rise to adrenergic innervation on cerebral vessels, induces a cerebral vasoconstriction; this is in agreement with the hypothesis of the involvement of a specific intrinsic neuronal pathway. Regarding a cholinergic mechanism, our results suggest that cholinergic receptors are also partly involved in the cerebrovascular reaction studied. Thus the response may be partly mediated by the parasympathetic cholinergic (cerebral) dilator pathways evidenced by several g r o u p s 9,12,2U,26 o r by central cholinergic pathways, for example an intrinsic system originating or projecting through the fastigial nucleus such as suggested by Reis et al? 3. These authors, on stimulating the fastigial nucleus, obtained an increase in BP and a much more pronounced increase in CBF than the increase in glucose consumption would have predicted. This finding apparently involving a cholinergic pathway, can perhaps be related to the results obtained by Pearce et al, 29 who concluded that a short-latency mechanism involving a cholinergic link may mediate cerebral vasodilatation during spontaneous E E G shifts. However. the phenomenon they studied is not exactly similar since
313 it occurs concomitantly with a decrease in blood pres-
the pO 2 increase during stress was significantly lower
sure.
than during control stress, although the C B F increase
The present study performed on immobilized rabbits strongly suggests that neurogenic mechanisms intervene in the regulation of cerebral blood flow in
was higher than that observed in the absence of the blocker. It seems that, as proposed by Siesj640 in the
physiological conditions which may correspond in hu-
an homeostatic mechanism maintaining oxygen availability, so that hypoxia of the cerebral tissue is
man beings to anxiety or recovery after surgery. However, the results also reveal an increase in local
case of hypoxic hypoxia, the hyperaemia represents
avoided during stress. The absence of significant var-
metabolism in this situation, partly responsible for the vasodilatation observed. If we consider the local
iations in p C O 2 can be explained by the very efficient
variations in pO 2 we can see that during the stress reaction induced without any previous injection of re-
In conclusion, the present study of immobilization stress in rabbits provides evidence of the involvement
ceptor blocker, pO2 increased because of the hyper-
of fl-adrenergic and cholinergic receptors and of local
aemia; however, when the increase in flow was diminished by propanolol or atropine, pO 2 decreased with respect to baseline levels. It seems clear that
metabolism in the cerebral vasodilatation which oc-
during the maximal vasodilatation, the high blood flow brought additional available oxygen which more
buffering of CO 2.
curs. It suggests that neurogenic mechanisms, probably intrinsic to brain, intervene in the stress reaction inducing an hyperaemia thus preventing energy failure in brain tissue.
than compensated the increase in metabolism, but the much weaker increase in blood flow after receptor blockade could not ensure the oxygen needs of
ACKNOWLEDGEMENTS
the tissue without a fall in pO2, i.e., more oxygen was consumed than blood flow could provide. O n the oth-
Thanks are due to Mr. J e a n - L o u p Correze for technical assistance, to Mrs. J e a n n e Leizerovici for
er hand, when a-adrenergic receptors were blocked,
typing the manuscript and to Dr. Richard Sercombe for criticism of the manuscript.
REFERENCES 1 Aubineau, P. F., Seylaz, J., Sercombe, R. and Mamo, H., Evidence for regional differences in the effect of beta-adrenergic stimulation on cerebral blood flow, Brain Research, 61 (1973) 153-161. 2 Aubineau, P. F., Sercombe, R. and Seylaz, J., Cholinergic influence on local cerebral blood flow in the rabbit by a direct vasodilator action and an inhibition of the sympatheticmediated vasoconstriction, Brit. J. Pharrnacol., 68 (1980) 449-459. 3 Belova, T. I. and Jonsson, G., Blood-brain barrier permeability and immobilization stress, Acta physiol, scand., 116 (1982) 21-29, 4 Carlsson, C., Hfigerdal, M. and Siesj6, B. K., Increase in cerebral oxygen uptake and blood flow in immobilization stress, Actaphysiol. scand., 95 (1975) 206-208. 5 Carlsson, C., Hfigerdal, M., Kaasik, A. E. and Siesj6, B. K., A catecholamine-mediated increase in cerebral oxygen uptake during immobilization stress in rats, Brain Research, 199 (1977) 223-231. 6 Dahlgren, N., Ingvar, M. and Siesj0, B. K., Effect of propranolol on local cerebral blood flow under normocapnic and hypercapnic conditions, J. Cereb. Blood Flow Metabol., 1 (1981) 429-436. 7 D'Alecy, L. G. and Rose, C. J., Parasympathetic cholinergic control of cerebral blood flow in dogs, Circulat. Res., 41 (1977) 324-331.
8 Edvinsson, L., Nielsen, K. C., Owman, Ch. and West, K. A., Alterations in intracranial pressure, blood brain barrier and brain edema after subchronic implantation of a canula into the brain of conscious animals, A cta physiol, scan&, 82 (1971) 527-531. 9 Edvinsson, L., Nielsen, K. C., Owman, Ch. and Sporrong, B., Cholinergic mechanisms in pial vessels. Histochemistry, electron microscopy and pharmacology, Z. Zellforsch., 134 (1972) 311-325. 10 Edvinsson, L. and Owman, Ch., Pharmacological characterization of adrenergic alpha and beta receptors mediating vasomotor response of cerebral arteries in vitro, Circulat. Res., 35 (1974) 835-849. 11 Edvinsson, L., Falck, B. and Owman, Ch., Possibilities for cholinergic action on smooth musculature and on sympathetic axons in brain vessels mediated by muscarinic and nicotinic receptors, J. Pharmacol. exp. Ther.. 200 (1977) 117-126. 12 Florence, V. M. and Bevan, J. A., Biochemical determination of cholinergic innervation in cerebral arteries, Circulat. Res., 45 (1979) 217-218. 13 Ingvar, D. H. and S6derberg, U., A new method for measuring cerebral blood flow in relation to the electroencephalogram, Electroenceph. clin. Neurophysiol., 8 (1956) 403-412. 14 Ingvar, D. H. and S6derberg, U., Cortical blood flow related to EEG patterns evoked by stimulation of brain stem, Acta physiol, scand., 42 (1958) 130-143.
314 15 Iwayama, T., Furness, J. B. and Burnstock, G., Dual adrenergic and cholinergic innervation of the cerebral arteries of the rat, Circular. Res., 26 (1970) 635-646. 16 Kety, S. S., Circulation and metabolism of the human brain in health and disease, Amer. J. Med., 8 (1950) 205-217. 17 King, B. D., Sokoloff, L. and Wechsler, R. L., The effects of t.-epinephrine and L-norepinephrine upon cerebral circulation and metabolism in man, J. clin. Invest., 31 11952) 273-279. 18 Lacombe, P., Reynier-Rebuffel. A. M. and Seylaz, J., Cerebral uptake of circulating catecholamines during stressinduced acute hypertension. In D. D. Heistad and M. L. Marcus (Eds.), Cerebral Blood Flow: Effects of Nerves and Neurotransmitters. Elsevier, New York. 1982, pp. 343-348. 19 Lacombe, P. and Seylaz, J., Significance of the cerebrovascular effects of immobilization stress in the rabbit, J. Cereb. Blood Flow Metabol., in press. 20 Lavrentieva. N. B., Mchedlishvili, G. I. and Plechkova, E. K., Distribution and activity of cholinesterase in the nervous structures of the pial arteries (a histochemical study), ByulL eksp. Biol. Med., 64 (1968) 110-113. 21 Leniger-Follert, E., Wrabetz, W. and Liibbers, D. W., Local tissue pO 2 and microflow of the brain cortex under varying arterial oxygen pressure. In J. Grote, D. Reneau and G. Thews (Eds.), Oxygen Transport to Tissue, Vol. 75, Plenum Press, New York. 1976, pp. 361-367. 22 Meyer, J. S., Nomura, F., Sakamoto, K. and Kondo, A., Effect of stimulation of the brain stem reticular formation on cerebral blood flow and oxygen consumption, Electroenceph. clin. Neurophysiol., 26 11969) 125-I32. 23 Meyer, J. S., Teraura, T., Sakamoto, K~ and Kondo, A., Central neurogenic control of cerebral blood flow, Neurology. 21 (1971) 247-263. 24 Nelson, E. and Rennels, M., Neuromuscular contacts in intracranial arteries of the cat, Science, 167 (1969) 301-302. 25 Nielsen, K. C. and Owman, Ch., Adrenergic innervation of pial arteries related to the circle of Willis in the cat, Brain Research, 6 (1967) 773-776. 26 Nielsen, K. C., Owman, Ch. and Sporrong, B., Ultrastructure of the autonomic innervation apparatus in the main pial arteries of rats and cats, Brain Research, 27 (1971) 25-32. 27 Ohata, M., Fredericks, W. R., Sundaram, U. and Rapoport, S. I.. Effects of immobilization stress on regional cerebral blood flow in the conscious rat, J. Cereb. Blood Flow Metabol., 1 (1981) 187-194. 28 Ohno, K., Pettigrew, K. D. and Rapoport, S. I., Lower limits of cerebrovascular permeability to non electrolytes in the conscious rat, Amer. J. Physiol., 235 (1978) H299-H307. 29 Pearce, W. J., Scremin, O. U., Sonnenschein, R. R. and Rubinstein, E. H., The electroencephalogram, blood flow and oxygen uptake in rabbit cerebrum, J. Cereb. Blood
Flow Metabol.. 1 ( 1981 ) 419-428. 30 Peperkamp. J. P. C.. Cerebral Blood Flon m the Rabbit. Normal Values and the Effects of Some Anesthetics and Vasoactive Substances on the Central Blood Flow. In: l"hesis in Neurobiology: Collected Reprints. Vol. 10. Centra] Institute for Brain Research. Amsterdam. 1972. pp. 1- 140. 31 Pinard. E. and Sevlaz. J.. Intracerebral gas partial pressures changes under vasoactive drugs: a mass spectrometry study, Pfliigers Arch. ges. Physiol., 375 (1978) 25-3t;. 32 Pinard. E.. Purves. M J.. Seylaz. J. and Vasquez. J.. The cholinergic pathway to cerebral blood vessels. II Physiok)gical studies. Pfli~gers Arch. ees. PhysioL, 379 /1979~ 165-172. 33 Reis. D. J., ladecola. C.. MacKenzie. E. T.. Mori. M.. Nakai. M. and Tucker. L. W.. Primary and metabolically coupled cerebrovascular dilation elicited by stimulation of two intrinsic systems of brain. In D. D. Heistad and M. k Marcus (Eds.), Cerebral Blood Flow: Effects of Nerves and Neurotransmuters Elsevier. New York_ 1982. pp. 475-484. 34 Scremin. O. U.. Rubinstein. E. H. and Sonnenschein. R. R. Cholinerglc modulation of the cerebrovascular response to CO z in the rabbit. In Ch. Owman and L. Edvinsson (Eds.), Neurogenic Control c~f the Brain Circulation Pergamon, New York. 1977. pp. 471-481. 35 Sercombe. R.. Aubineau. P.. Edvinsson. L.. Mamo. H.. Owman. Ch.. Pinard. E. and Seylaz. ,I.. Neurogenic influence on local cerebral blood flow. Neurology, 25 (1975] 954-963. 36 Sercombe. R.. Lacombe. P.. Aubineau, P . Mamo. H.. Pinard. E.. Reynier-Rebuffel. A. M. and Seylaz. J.. Is there an active mechanism limiting the influence of the sympathetic system on the cerebral vascular bed? Evidence for vasomotor escape from sympathetic stimulation in the rabbit, Brain Research, 164 (1979) 81-102. 37 Seylaz, J., Aubineau, P. F., Correze, J. L. and Mamo, H., Techniques for continuous measurement of local cerebral blood flow, PaO2, PaCO2 and blood pressure in the non anesthetized animal, Pfliigers Arch. ges. Physiol.. 340 (1973) 175-180. 38 Seylaz, J., Pinard, E., Correze, J. L., Aubineau, P. F. and Mamo, H., Quantitative continuous measurement of blood gas tensions by mass spectrometry, J. appl. Physiol., 37 (1974) 937-941. 39 Seylaz, J. and Pinard, E., Continuous measurement of gas partial pressures in intracerebrat tissue, J. appl. Physiol., 4411978) 528-533. 40 Siesjo, B. K., Brain Energy Metabolism, Wiley, New York, 1978, 285 and 422 pp. 41 Sokoloff, L., Cerebral circulation and behavior in man: strategy and findings. In A. J. Mandell and M. P. Mandell (Eds.), Psychochemical Research in Man, Academic, New York, 1969, pp. 237-252.