J Neurosurg 50:441-448, 1979

Cerebrovascular responses to subarachnoid blood and serotonin in the monkey DONALD P. J. BOISVERT,M.D., BRYCE K. A. WEIR, M.D., THOMAS R. OVERTON,PH.D., RHODERICK J. REIFFENSTEIN, PH.D., AND MICHAEL G. A. GRACE, PH.D. Divisions of Neurosurgery and Biomedical Engineering, and Department of Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada u,' Preliminary in vitro experiments were performed to determine the serum concentration of serotonin in the monkey, and the ability of cyproheptadine to block serotonin and serum-induced contractions in monkey cerebral arteries. Thirty-four cynomolgus monkeys were subsequently used to study changes in regional cerebral blood flow (CBF) obtained by the intracarotid x88Xetechnique, and in the angiographic cerebral arterial caliber resulting from subarachnoid injection of artificial cerebrospinal fluid (CSF), blood, and serotonin. Five animals in each injection group were given 1.0 mg/kg intravenous cyproheptadine (a serotonin-blocking agent) during the post-injection period. Subarachnoid injection of artificial CSF produced no change in CBF or arterial caliber. Post-injection administration of cyproheptadine also had no effect on these parameters. A subarachnoid injection of fresh autogenous blood produced a significant but transient (less than 1 hour) decrease in CBF and moderate vasospasm, which lasted at least 3 hours. This vasospasm was essentially unaffected by intravenous cyproheptadine. The CBF and arterial caliber were unchanged following a subarachnoid injection of serotonin at concentrations (5 • 10-6M)present in normal monkey serum. In contrast, 5 • 10 o M serotonin invariably produced near maximal contractions in the in vitro cerebral artery preparations. Higher (• 10) serotonin concentrations caused a transient CBF response similar to that obtained with blood. However, the cerebral vasospasm induced was of shorter duration than that obtained with blood. These results do not support a major role for serotonin in the production of post-subarachnoid hemorrhage vasospasm. Moreover, our data indicate that in vitro experiments do not reflect the ability of serotonin to constrict cerebral arteries in the intact animal. KEY WORDS serotonin 9 subarachnoid hemorrhage cerebral vasospasm 9 cynomolgus monkey 9

ECAUSE of widespread belief that cerebral vasospasm is a major factor contributing to the development of cerebral ischemia in patients with subarachnoid hemorrhage (SAH), intensive research has been carried out in an attempt to identify the factor(s) in blood that are capable of producing prolonged constriction of the cerebral arteries. At present, the bulk of accumulated evidence points toward a chemical vasoconstrictive agent that is liberated from the blood present in the subarachnoid space. Many such agents have been identified in blood (for instance, serotonin, prostaglandins, norepinephrine), and each has its proponents as the agent causing cerebral vasospasm in man. However, recent evidence, particularly from in vitro studies, appears to favor serotonin as the major spasmogenic agent. 1-4'18'18 The present study was designed to assess the spasmogenic ability of serotonin in a primate model, to determine the relationship between serotonin-

B

J. Neurosurg. / Volume 50 / April, 1979

9 cerebral blood flow

9

induced vasospasm and cerebral blood flow (CBF), and to compare the results to those obtained under the same conditions with artificial cerebrospinal fluid (CSF) and blood. Materials and Methods

In Vitro Experiments In vitro experiments were carried out to obtain an estimate of the normal concentration of serum serotonin in monkey serum. The results of these experiments determined the concentration of the serotonin solution to be injected into the subarachnoid space in the experiments in vivo. A second set of experiments in vitro was conducted to verify the ability of cyproheptadine to block contractions in monkey cerebral arteries induced by serotonin and serum. Tissues known to be reactive to serotonin (Table 1) were placed into cooled, oxygenated, modified Krebs44"1

D. P. J. B o i s v e r t , et al. Henseliet solution after dissection. Spiral strips of these tissues were then cut and mounted in jacketed 5- or 10-ml organ baths maintained at 37 ~ C, and aerated with a 5% CO2/95% gas mixture (pH -- 7.4). After a 2-hour recovery period, the response of the tissue to various agents (serotonin, cyproheptadine, monkey serum) was tested. Isometric tissue responses were measured using a force displacement transducer, connected to a Grass 4-channel polygraph (model 5D) or a Beckman dynograph.* The modified KrebsHenseliet solution bathing the tissues in the organ had the following composition: N a § 117.5 mM; Ca ++, 2.5 mM; CI-, 127.9 mM; POi s, 1.2 mM; K +, 5.4 mM; Mg §2470.6 mM; SOt, 0.6 mM; glucose, 11.1 m M . Monkey serum serotonin concentrations were estimated by comparing serum-induced contractions to contractions obtained using known concentrations of serotonin. A minimum of two concentrations of serotonin and two concentrations of serum was used for each serum serotonin determination (four-point assay). In other experiments in vitro, the response of the tissue (monkey cerebral arteries) to cumulative concentrations of serotonin or specific concentrations (5 X 10 -6 M) of serotonin and serum were determined in the presence and absence of the serotonin antagonist cyproheptadine at a concentration of 5 X 10-6 M. In Vivo Experiments Thirty-four female cynomolgus (Macaca irus) monkeys weighing 2.3 to 4.2 kg were used for the study. Anesthesia for the surgical preparation was achieved with intravenous sodium pentothal (25 to 30 mg/kg). Light general anesthesia was subsequently maintained with a mixture of nitrous oxide and oxygen from a reservoir in a ratio of 2: 1. The animals were ventilated with a H a r v a r d variable-phase respiratort and paralyzed with intravenous gallamine for the duration of the experiment. Body temperature was continuously monitored by an esophageal thermometer,~ and maintained between 36 ~ and 38 ~ C by a small heating pad positioned below the animals. Standard lead electrocardiography (EKG) was performed in all animals. Femoral artery catheterization was performed in the anesthetized animals, and arterial blood samples *Grass Model FT force displacement transducer and Grass 4-channel polygraph manufactured by Grass Instrument Company, Quincy, Massachusetts. Beckman dynograph Model B manufactured by Beckman Instruments, Inc., 2500 Harbor Boulevard, Fullerton, California. +Harvard variable-phase respirator manufactured by Harvard Apparatus, Inc., Dover Road, Millis, Massachusetts. SEsophageal thermometer (Tele-Thermometer) manufactured by Yellow Springs Instrument Company, Yellow Springs, Ohio. 442

TABLE 1 Bioassay of monkey serum serotonin concentrations Monkey No.*

Tissue

R-1 umbilicalartery R-2 basilar artery (dog) R-2 middle cerebral artery (dog) R-3 umbilical artery R-3 umbilical artery R-3 umbilical artery R-4 basilar artery (dog) R-4 umbilicalartery R-4 umbilical artery R-5 umbilical artery mean C-1 umbilicalartery C-2 umbilical artery C-3 umbilical artery C-4 umbilical artery C-5 umbilical artery C-6 stomach fundus (rat) C-6 stomach fundus C-7 stomach fundus C-7 stomach fundus mean overall mean

Molar Concentration of Serum 5-HT 2.5 2.0 3.5 6.2 5.3 1.2 2.3 8.4

X X X X X X X X

2.8 3.6 3.8 3.0

X • X X

1.4 X 10-5 10-6 2.2 X 10-6 10-6 10-6 10-G 5.0 X 10-6 10-6 10-~ 10-6 7.6 X 10-6 10-6 1.1 X 10-6 8.0 • 10-6 6.9 X 10-6 6.3 X 10-6 1.1 X 10-5 3.4 X 10-6 1.7 X 10-5 10-6 10-6 10-8 10-6 3.4 X 10-6 1.6 • 10-5 1.3 X 10-6

*R = rhesus monkeys; C = cynomolgus monkeys.

were immediately obtained for p H and blood gas analysis.w By adjusting the volume output from the respirator, arterial pH, carbon dioxide (PaCO~), and oxygen (PaO2) values were kept within the physiological range during surgery. Thereafter, analysis of arterial pH and blood gas was performed during each measurement of CBF. Hematocrit determinations, required for CBF calculation, were carried out four to six times in the course of each experiment. Mean arterial blood pressure (MABP) was continuously monitored by a pressure transducer ]l connected to the femoral artery catheter. Cerebrospinal fluid pressure was also continuously monitored by a similar pressure transducer connected to a catheter positioned in the lumbar subarachnoid space. A cranial twist-drill hole (1.5 m m diameter) was made 0.5 to 1.0 cm dorsal to the nasion in the animals to be subjected to subarachnoid injections. Hemostasis was achieved with bone wax, and the defect was sealed until the time of subarachnoid injection. Cervical dissection to the common carotid artery bifurcation was p e r f o r m e d with the aid of an operating microscope. A 7.0 silk purse-string suture was then placed in the wall of the common carotid w gas analyzer, Model 113, manufactured by Instrumentation Laboratory, Inc., 133 Hartwell Avenue, Lexington, Massachusetts. II Statham P23dB pressure transducer manufactured by Statham Instruments Company, 2230 Statham Boulevard, Oxnard, California.

J. Neurosurg. / Volume 50 / April, I979

Cerebrovaseular response to blood and serotonin artery approximately 2 cm proximal to the bifurcation; the diameter of the circle formed by the pursestring suture was approximately 1 ram. A No. 20 catheter was inserted into the common carotid artery through the space enclosed by the suture and threaded to the origin of the internal carotid artery (ICA). The external carotid artery was clipped at its origin using a modified aneurysm clip. The carotid artery catheter was then connected to an injector device via a No. 16 catheter and three-way stopcock assembly. At the end of the experiment the carotid catheter was removed, and the purse-string suture tightened to close the small arteriotomy. Before subarachnoid injection, a circumferentially bevelled, No. 19 spinal needle was inserted (under fluoroscopy) along the floor of the anterior fossa into the chiasmatic cistern. Adequate placement of the needle was confirmed with return of CSF. The needle was then secured to the skull by a screw device. Three ml of fresh autogenous arterial blood, artificial CSF, 6 or serotonin solution was injected into the subarachnoid space within approximately 25 seconds. In all experiments, artificial CSF was freshly prepared from stock solutions just before SAH. The serotonin solution was prepared by mixing a small volume (0.5 or 5 ml) of serotonin stock solution (1 X 10-3 M) into 100 ml of artificial CSF to produce a final serotonin concentration of 5 X 10 ~ M or 5 X 10 5 M. All solutions were adjusted to pH 7.35 and to a temperature of 36 ~ to 38 ~ C before subarachnoid injection. Experimental Design The experimental design for the in vivo experiments consisted of three major experimental categories that were established on the basis of the type of subarachnoid injection to be performed; blood, serotonin, and artificial CSF. Each major category was then subdivided into one group that would receive cyproheptadine and one group that would not receive it: Group 1: SAH Group 2: SAH followed by serotonin blocking agent (intravenous cyproheptadine, 1.0 mg/kg) Group 3: Subarachnoid serotonin injection Group 4: Subarachnoid serotonin injection followed by serotonin blocking agent Group 5: Subarachnoid artificial CSF injection (mock SAH) Group 6: Mock SAH followed by serotonin blocking agent (intravenous cyproheptadine, 1.0 mg/kg). These treatment categories were assigned to randomized blocks; each randomized block containing the six treatment categories in random order. Experiments were then performed according to this design. A total of 30 monkeys were utilized in this series of experiments, each treatment group containing five animals. J. Neurosurg. / Volume 50 / April, 1979

The major variables measured were rCBF, cerebral vessel caliber, and cerebral perfusion pressure ( M A B P - CSF pressure). Secondary variables measured included PaCO~, PaO~, pH, heart rate, and hematocrit. All statistical comparisons of pre- and post-subarachnoid injection values were done using the Student's t-test. After completion of the above experiments, a tenfold increase of the serotonin concentration was injected into the subarachnoid space of an additional four monkeys. The experimental protocol was slightly different for these animals in that cerebral angiograpby was also performed immediately following subarachnoid injection and only two to three rCBF measurements were made before taking the final angiograms. These animals did not receive cyproheptadine. Regional CBF and Vessel Caliber Measurements Cerebral blood flow was measured from four cerebral regions and the orbitomaxillary and cerebellar regions as previously described. 11 However, in the present study, an automatic injector system was used to inject lS3Xe into the ICA? 4 Cerebral arterial caliber measurements were obtained from lateral angiograms as previously described. Changes in CBF, u whether spontaneous or induced by subarachnoid injection, invariably occurred simultaneously and almost equally in all four cerebral regions monitored. Therefore, CBF values used for analysis represent the mean of the four cerebral regions. In all cases, the CBF values were determined using the height/area method of analysis. The intradural ICA was the most consistently well visualized on lateral angiograms. Therefore, caliber measurements from this site were used for most of the data analysis. Results

Experiments In Vitro Values for the serotonin bioassay determinations are given in Table 1. The mean serotonin concentration value (1.6 X 10 5 M) for cynomolgus monkey serum was higher than the mean value for rhesus monkey serum. This discrepancy, however, was due to a very high value obtained from one cynomolgus monkey (Monkey C-l). The mean concentration value obtained for cynomolgus monkey serum is 7.4 x 10 6 M when Monkey C-1 is excluded. The median value is 6.3 • 10.8 M. Most of the values were between 2.0 and 8.5 x 10-8 M. On the basis of these results, a concentration of 5 X 10 6 M for serotonin was defined as a physiological concentration. Thus, in subsequent in vivo experiments a solution (artificial CSF) containing 5 x 10-6 M serotonin was used for subarachnoid injections (with the exception of a separate group of four animals in which 5 X 10 5 M serotonin was used.) 443

D. P. J. Boisvert, et al. r

cA

1OOpI

S-HT

CH F2~

Ser um~

FIG. 1. In vitro blocking by cyproheptadine (CH) of contractions induced by serotonin (5-HT) and monkey serum. Response of monkey internal carotid artery (ICA) and basilar artery (BA). F2~ = prostaglandin F~.

A s e c o n d series o f in vitro e x p e r i m e n t s was c o n d u c t e d in w h i c h c y p r o h e p t a d i n e was used to b l o c k s e r o t o n i n - a n d s e r u m - i n d u c e d c o n t r a c t i o n s in t h e I C A and b a s i l a r a r t e r i e s o f c y n o m o l g u s m o n k e y s . C o n t r a c tions i n d u c e d by s e r u m a n d s e r o t o n i n (5 X 10 -8 M ) w e r e c o m p l e t e l y b l o c k e d by 5 X 10 8 M c y p r o h e p t a d i n e (Fig. 1). S u b s e q u e n t i n j e c t i o n o f t h e p r o s t a g l a n din F ~ ( P G F 2 , ) into t h e o r g a n b a t h d e m o n s t r a t e d t h a t the a r t e r i a l p r e p a r a t i o n s w e r e still c a p a b l e o f v i g o r o u s contraction. Serotonin dose-response curves were d e t e r m i n e d w i t h a n d w i t h o u t c y p r o h e p t a d i n e in t w o vessels. C y p r o h e p t a d i n e (5 X 10 -6 M ) c o m p l e t e l y b l o c k e d t h e r e s p o n s e to s e r o t o n i n in b o t h t h e I C A a n d b a s i l a r a r t e r y . In five o t h e r I C A a n d f o u r b a s i l a r artery preparations, the percentage maximal contract i o n w i t h 5 • 10 -6 M s e r o t o n i n was 84.3% • 16.2

( S D ) and 93.5% • 6.2, r e s p e c t i v e l y . I n t h e p r e s e n c e o f 5 x 10 -6 M c y p r o h e p t a d i n e , t h e c o r r e s p o n d i n g perc e n t a g e v a l u e s w e r e 2.3% + 3.6 a n d 0.2% + 0.5.

Experiments I n V i v o Physiological data values are presented according to t r e a t m e n t in T a b l e 2. T h e s e d a t a a r e f u r t h e r subd i v i d e d into t h r e e g r o u p i n g s : 1) p r e - s u b a r a c h n o i d inj e c t i o n d a t a , 2) p o s t - s u b a r a c h n o i d i n j e c t i o n d a t a obt a i n e d b e f o r e t h e first p o s t - i n j e c t i o n a n g i o g r a m s , a n d 3) p o s t - s u b a r a c h n o i d i n j e c t i o n d a t a o b t a i n e d following t h e first p o s t - i n j e c t i o n a n g i o g r a m s or i n t r a v e n o u s administration of cyproheptadine. The mean pH values for t h e s e g r o u p s r a n g e d f r o m 7.38 to 7.44.

TABLE 2

Physiological data in six treatment groups* Values

Group 1

PaCO2 pre-SSI 41 post-SSI 38 post-SSI -F CH 39 PaO~ pre-SSI 135 post-SSI 134 post-SSI q- CH 135 mean arterial blood pressure pre-SSI 110 post-SSI 108 post-SSI + CH 106 eerebrospinal fluid pressure pre-SSI 10 post-SSI 18 post-SSI+CH 19

Group 2

Group 3

Group 4

Group 5

Group 6

• 1 • 1 • 1

41 =~ 1 40 + 1 39 • 0

41 • 2 40 • 0 40 • 1

42 ~ 2 39 :~ 1 40 • 2

41 • 1 39 • 1 39 • 1

40 ~: 2 38 • 1 38 =~ 1

• 2 ~= 1 =~ 1

129 =~ 2 135 • 6 141 e= 14

128 • 3 130 • 0 127 ~= 1

125 • 2 131 • 5 128 • 3

130 • 4 132 ~: 3 132 • 7

134 • 3 134 • 2 132 • 2

• 4 =~ 4 • 3

108 :a: 2 112 • 4 119 • 4

112 :~ 6 110 • 1 107 • 4

106 • 1 102 • 3 107 • 3

110 • 5 109 • 5 109 ~: 6

107 • 3 107 • 3 102 • 2

• 0 :a: 2 • 1

8 • 0 14 • 1 19 • 1

9 • 1 11 • I l0 • 1

11 • 0 10 • 0 12 • 2

10:~ 1 12 • 3 13 • 4

9 • 1 11 • 1 10 • 1

*All values are in mm Hg, mean :~ SD. For treatment groups see text. tPre-SSI = before subaracbnoid serotonin injection (SSI); post-SSI = after SSI but before post-SS! angiograms; post-SSI + CH = after SSI and the first post-SSI angiograms or intravenous administration of cyproheptadine. 444

J. Neurosurg. / Volume 50 / April, 1979

Cerebrovascular response to blood and serotonin

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Cerebrovascular responses to subarachnoid blood and serotonin in the monkey.

J Neurosurg 50:441-448, 1979 Cerebrovascular responses to subarachnoid blood and serotonin in the monkey DONALD P. J. BOISVERT,M.D., BRYCE K. A. WEIR...
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