Constriction of Pial Arterioles Produced by Prostaglandin BY WILLIAM I. ROSENBLUM, M.O. Abstract: Constriction of Pial Arterioles Produced by Prostaglandin F2U
• Prostaglandin F 2a constricted pial arterioles when locally applied to the cerebral surface. Norepinephrine and serotonin each elicited similar contractile effects. The constriction produced by F 2o in combination with either biogenic amine was greater than the constriction elicited by F2a or amine acting alone. The effect of one agent on the other was additive rather than potentiating. Since F2o, norepinephrine and serotonin are all naturally occurring agents, it is possible that their combined effect is important under pathological circumstances and this combined effect should not be overlooked in the search for single spasmogens of great potency. Before ascribing a pathologically important effect to F 2a , either alone or in combination, evidence is required showing that doses effective in experiments are similar to the concentrations occurring during disease states and/or that vessels may become hypersensitive to F 2a during such states.
Additional Key Words norepinephrine
• Prostaglandins (PG) are a group of 20-carbon, unsaturated lipid acids with a wide variety of possible biological actions, some of which are just beginning to be explored.1"3 They have been found to produce constriction or relaxation of smooth muscle, including vascular smooth muscle, in vitro and in vivo. The direction of response is dependent upon the type of PG and the test object. Similarly, they have been reported to facilitate or to inhibit adrenergic responses, sometimes by acting on the release of norepinephrine and sometimes by altering the responsivity of the test organ. Prior to this writing, the author is aware of only four reports describing the effect of the PG, known as F2n, on cerebral blood vessels. The results of this small number of studies suggested that additional investigation was warranted. Prior reports indicated that F2ct constricts blood vessels somewhere in the cerebral vasculature. 46 Two dealt, at least partly, with surface arteries and arterioles. One of these showed that intracarotid injection caused constriction of pial arteries and arterioles under 200 n in diameter, 6 while the other 5 demonstrated that intracisternal F2Q produced constriction of large surface arteries. These recent investigations suggested that the action of F 2a might be relevant to the clinical problem of vasospasm, especially since prostaglandins, including F 2o , are present in brain and released by brain into the cerebrospinal fluid (CSF). 7 9 A relationship between PGs and clinical vasospasm also is suggested by the fact that platelets release PGs,10 and a platelet facProfessor and Chairman, Division of Neuropathology, Medical College of Virginia, Box 17, Health Sciences Center, Richmond, Virginia 23298. Supported by HL 12775 and the John A. Hartford Foundation.
microcirculation serotonin
cerebral blood vessels vasospasm
tor(s) has been implicated in the vasospasm associated with subarachnoid hemorrhage. The present investigation focuses attention on the response of smaller pial arterioles, after local application of F 2a to the cerebral (i.e., arachnoid) surface. Under such circumstances, the PG will arrive at cerebral surface vessels (pial vessels) after crossing the arachnoid and passing through the CSF. Transport through the CSF also represents a route by which they would arrive at the cerebral blood vessels after release from brain or from platelets in the subarachnoid space. After initiating our studies, an additional report appeared11 describing constriction of pial arterioles and small arteries in the cat following application of F 2o to the cerebral surface. The technique used therein more closely resembles our own than do those in any of the earlier studies.
Methods Male mice, Swiss strain (Dublin Farms), were used. These weighed 18 to 35 gm and were anesthetized with urethane. A tracheotomy and craniotomy were performed and the dura stripped from the craniotomy site. The brain surface with the exposed pial vessels covered only by the transparent arachnoid was then irrigated continuously with a mock cerebrospinal fluid (Elliott's solution). This solution has the following composition: Na + 134 mEq/1; C a + + 2.7 mEq/1; M g + + 2 . 4 mEq/1; Cl 137 mEq/1; SO", 2.4 mEq/1; HPO< 1.5 mEq/1, and dextrose 800 mg/1. In addition the solution contains initially 2.3 mEq/1 HCO3, and is acidic. The pH rises on standing or mild heating and is readjusted to physiological levels (between 7.3 and 7.4) by bubbling CO 2 through the solution. This solution was delivered to the brain surface at the rate of 2 ml per minute by a constant perfusion pump which caused the fluid to pass through a heating coil and hypodermic needle. The orifice of the needle was a few millimeters
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above the craniotomy site and solutions emanating from the needle orifice fell in a continuous series of drops to the edge of the craniotomy and spread in a thin film over the cerebral surface, from whence the fluid was led by a cotton wick. The temperature of the fluid at the craniotomy site was 37°C. The pH of the fluid was monitored repeatedly during the observation of each mouse in the study, by sampling drops from the needle orifice. The pH of more than 100 measurements was 7.34 ± 0.03 (M ± SD). A side arm just proximal to the heating coil permitted delivery of a 1 ml test solution as a bolus interrupting the normal irrigant. This bolus took 2 minutes ± 30 seconds to reach the cerebral surface, at which time its temperature was the same as that of the normal irrigant. The pH of the bolus and that of the irrigant were within 0.05 pH units of each other. The bolus cleared the brain surface in 30 seconds, after which the regular irrigant was once again flowing over the brain. Solutions of 5% BaCl2 were used to test the reactivity of the pial arterioles before and after each experiment.12 All animals entering the study responded to the barium ion with an arteriolar constriction both before and after the application of F2(t. The prostaglandin F2Q used in this study was the tromethamine (THAM) salt, with a molecular weight of 475.6. The free acid has a molecular weight of 354.5. THAM, when used by itself over a dose range exceeding that applied in the present investigation (10" 5 -10 3 M), failed to alter the diameter of the pial arterioles. Norepinephrine (NOR) was used as the bitartrate and serotonin (5HT) as the creatinine sulfate. Fresh solutions of all agents were made each day in the Elliott solution and the pH adjusted to between 7.3 and 7.4 by adjusting the CO 2 in the solution. Preliminary studies with F2f, alone or combined with NOR showed no change in blood pressure during the local application of these agents to the pial surface and for five minutes thereafter. Thus during the 30-second period of application insignificant amounts of drug were absorbed into the blood stream. The internal diameter of the pial arterioles was measured by using a microscope, image splitter and TV monitor as described by Baez.13- " The internal diameter of the vessels was represented by a dark column of red cells and thus was underestimated by approximately 4 jt.15 The diameter of the vessels was monitored for five minutes prior to the time the test solution hit the brain, and continued to be monitored for eight minutes thereafter. Stability of baseline diameter prior to application of test solution was required and animals not showing such stability were discarded. If a change in diameter occurred within the 60 seconds following arrival of test solution at the cerebral surface, the change was counted as a response. In practice, over
90% of the responses occurred during the first 30 seconds. The point of maximal change in diameter was used to quantify the response. In these experiments the limiting factor in measuring diameter was the reproducibility with which successive measurements of the same vessel could be made. This depends upon the observer's ability to exactly juxtapose sheared images on the TV monitor and this, in turn, depends upon the clarity of the TV image and the contrast between vessel and background. In our system, which utilizes light reflected from the object being observed12'15 rather than transmitted light, we have tested our ability to make reproducible successive measurements of the distance between lines on a stage micrometer, a fixed distance demarcated by highly contrasted lines. These measurements never varied by more than 2 ju> and the reproducibility was independent of the distance between the lines over a range of 20 to 60 n. We took this 2 n discrepancy between successive measurements of a fixed object as the maximal "random" error of the method of measurement in our studies and arbitrarily discarded all changes smaller than 2 n from consideration as true changes. In other words changes of less than 2 n could not be distinguished from "noise." This change would equal approximately 4% of the mean resting diameter of arterioles in this study. Thus there will be a tendency to underestimate the frequency and magnitude of response to the vasoactive agents, especially at low dose levels.
Results EFFECTS OF SINGLE DOSES OF Fa,, ALONE
F 2a constricted pial arterioles as indicated in table 1. No relationship to dose could be seen until the lowest dose in the series, 10 ng per milliliter, where a marked decrement in response was observed. This decrement, however, was due solely to the failure of six of the ten animals to display any recognizable response, thus being counted as 0% constriction. In all other groups 70% to 100% of the animals responded to F 2a - Thus, in each group of ten mice, seven responded to 250 ng per milliliter, eight to 100 ng per milliliter, seven to 50 ng per milliliter, and all ten to 25 ng per milliliter. It is probable that the incidence of response was even higher, since small responses equal to less than 5% of resting diameter would have been dismissed as noise due to either spontaneous change in diameter or observer error in resetting the image splitter to the same null position between successive measurements.
TABLE 1
Effect of F, a Alone Dose F, a (fig/ml)* Number of experiments! Original diameter^ Percent constriction§
250 10 49 ± 15 11 ± 11
100 16 47 ± 12 14 ± 9
50 10 54 => = 13 12 ±9= 9
25 10 46 * 5 20 ± 7
10 10
45 ± 16 5 ± 10
*Administered as a 1 ml bolus over a 30-second period. fEquals number of mice. Only one dose per mouse per experiment. IMicrons =*= standard deviation. §Percent reduction from baseline diameter (mean =±= SD). 294
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PROSTAGLANDIN AND PIAL ARTERIOLES DOSE RESPONSE RELATIONSHIP TO F2(,
As indicated above, groups of animals failed to display a relationship between the average response of the group and the dose used in that group. We believe this was due to the great variability in the responses of the mice within each group. However, if successively larger doses were applied to the same animal, allowing the arteriole to recover from one dose before applying the next, greater constrictions were observed in the individual mouse as higher doses were applied to the pial surface of that mouse. This is shown in figure 1. The figure displays the results in five consecutive animals whose vascular reactivity was unaltered during the course of the experiment. The latter point was checked by comparing the response to BaCl2 before and after the series of F2«. In each animal the two responses to BaCl2 were within 10% of each other. INTERACTION OF Fto WITH SEROTONIN OR NOREPINEPHRINE
In each experiment an effective dose of amine was
EFFECT OF INCREASING DOSES OF F O O C
determined. This dose of amine was given alone, as was a dose of F 2a , and the response to each was measured. In addition, the same doses were combined and the response to the combination was measured. The order in which these drugs were given was varied, so that in some cases amine was given first, and in others the prostaglandin or the combination. The results were the same, irrespective of the order in which these agents were applied. The responses to F 2a were increased if 5HT or NOR were present at the same time. The response to the combination of drugs was equal to the sum of the responses to each of the components in the combination. The data are summarized in table 2, which displays the average response to the amine alone, to the F2a alone, and to the combination. In these experiments, the dose of NOR was either 0.5 or 5 ng per milliliter and the dose of 5HT was either 5, 10 or 20 jug per milliliter. The dose of F2Q was either 5, 25, or 50 ng per milliliter. The reader must remember that in each experiment the doses of amine and F 2o used singly were the same as those used in combination. The additive effect of NOR plus F 2Q or of 5HT plus F 2a was observed irrespective of the doses selected.
Discussion Our data clearly demonstrate constriction of pial arterioles after local application of F 2a . The results confirm the four previous reports7"9' u of which we are aware. The effective doses in our study are remarkably similar to those doses locally applied to the brain surface of the cat by Welch et al.11 in their study of the effect of F 2a on pial vessels. The latter is the only published study, of which we are aware, in which F 2Q was directly applied to the external surface of the brain and the pial vessels. Although we have not used duration of response as a parameter in this particular study, the response peak occurred between ten seconds and one minute following application of the drug, and thus the time course of the response was also similar to the data reported by Welch et al.11 These investigators did not report dose-response data, nor did they concern themselves with the interaction of F 2a and other vasoconstrictors. Our data show a simple, additive effect between TABLE 2
Interaction of F2a With Vasoactive Amines % Constriction*
80
FIGURE 1
Dose-response relationships are plotted for each of five mice. A single arteriole was monitored in each mouse, and the smallest diameter reached during the response was compared with the resting diameter. The decrement in diameter was expressed as a percent of the resting diameter.
Norepinephrine Prostaglandin F2a Combination Serotonin Prostaglandin Fna Combination
21 ± 8 22 ± 7 41 ± 14 13 ± 7 32 ± 10
N = 6f N = 6
42 ± 11
*Mean =•= standard deviation. Constriction as a percent of resting diameter. fN = number of experiments. 295
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the response to F2a and the response to NOR or 5HT. These results contrast with those reported for isolated extracerebral vessels16'" where F2a was shown to potentiate the responses to 5HT and other contractile stimuli rather than produce a simple additive response. However, in the latter experiments much lower concentrations of F2Q were used, concentrations which themselves had no effect on vascular tone, and of course cerebral vessels were not tested. Our own results may be of some interest, since they suggest that marked constriction of cerebral arterioles may follow contact with a combination of agents, any one of which might produce only modest constriction if acting alone. Thus the search for a single, elusive "spasmogen"18"20 to account for clinically important vasospasm may be missing an important point, namely the additive effect of several agents released by platelets and brain tissue into the CSF and subarachnoid space. One must make further mention of the arteriolar constriction induced by either NOR or 5HT in these experiments. In earlier publications by this author the contractile response of pial arterioles to NOR was described as unreliable12 or nonexistent21 and negative data also were reported with respect to a contractile effect of 5HT.22 More recently, we have reported a reliable, dose-related constriction elicited by NOR.23 This is confirmed in the present experiments, and a constriction induced by 5HT is also described. Several differences in methodology have been introduced since the earlier negative studies. These include better control of pH and temperature; a shift from pentobarbital to urethane anesthesia; avoidance of applying to the same preparation multiple doses of vasoactive drug in maximal strength; and the use of the TV microscope and image splitter to provide reliable, sensitive measurements of diameter, and a written record of diameter changes during the experiment. We cannot say which of these factors may have contributed to the change in our results. We have tried pentobarbital anesthesia in conjunction with the new recording system and we continue to get constriction like that observed with urethane. Thus far, the reliability of the recording system seems extremely important, since in the past small transient constrictions induced by single applications of NOR or 5HT might have been overlooked or dismissed as illusory when viewed by peering directly down the microscope aided only by an ocular micrometer. However, extremely small changes were consistently recorded with the more primitive technique when other vasoactive drugs were used.24(lable"
Whatever the explanation for our previous inconsistent or negative data with biogenic amines, the present studies have been markedly consistent, provide evidence not only for a contractile effect of these agents but also for F2Q, and indicate an additive effect of the latter with either NOR or 5HT. Unfortunately, in order to solidly implicate F2a as a contractile
stimulus of clinical importance, or for that matter to implicate NOR or 5HT, one must show that their concentrations in these and other experiments are comparable to that occurring in disease states. This is not the case, and in the present study, as well as the studies of other workers, each of these vasoactive agents is effective only in concentrations greater than those known to occur even during pathological conditions.25- 26 However, the concentration of these materials during pathological states is still relatively unexplored and for PG is virtually unknown with respect to the central nervous system and cerebrospinal fluid. Moreover, the sensitivity of pial vessels may be enhanced during periods of microcirculatory derangement.2123 Thus it will be important to test responses to F2Q alone and in the presence of other vasoactive agents, when such derangements are present, in order to see whether marked constriction can be elicited by doses lower than those used here.
Acknowledgments Ms. Mary Allgood and Mr. Guy Nelson provided invaluable technical assistance.
References 1. Bergstrom S, Carlson tD, Weeks JR: The prostaglandins: A family of biologically active lipids. Pharmacol Rev 20:1-48, 1968 2. McGiff JC, Crowshow K, Itskovitz HD: Prostaglandins and renal function. Fed Proc 33:39-47, 1974 3. Brady MJ, Kadowitz PJ: Prostaglandins as modulators of the autonomic nervous system. Fed Proc 33:48-60, 1974 4. White RP, Heaton JA, Denton IC: Pharmacologic comparison of prostaglandin F2o, serotonin and norepinephrine on cerebrovascular tone of monkey. Europ J Pharmacol 15:300-309, 1971 5. Pennink M, White RW, Crockarell J, et ah Role of prostaglandin F2(1 in the genesis of experimental cerebral vasospasm. Angiographic study in dogs. J Neurosurg 37:398-406, 1972 6. Yamamoto Yt, Feindel W, Wolfe tS, et ah Experimental vasoconstriction of cerebral arteries by prostaglandins. J Neurosurg 37:385-397, 1972 7. Holmes SW, Horton EW: The identification of four prostaglandins in dog brain and their regional distribution in the central nervous system. J Physiol 195:731-741, 1968 8. Bradley PB, Gilliam M, Samuels R, et ah Correlation of prostaglandin release from the cerebral cortex of cats with the electrocorticogram, following stimulation of the recticular formation. Brit J Pharmacol 37:151-157, 1969 9. Holmes SW: The spontaneous release of prostaglandins into the cerebral ventricles of the dog and the effect of external factors on this release. Brit J Pharmacol 38:653-656, 1970 10. Smith JB, Willis AL: Formation and release of prostaglandins by platelets in response to thrombin. Brit J Pharmacol 40:545-546, 1970 11. Welch KMA, Knowles t, Spira P: Local effect of prostaglandins on cat pial arteries. Europ J Pharmacol 25:155-158, 1974 12. Rosenblum W l , Zweifach BW: Cerebral microcirculation in the mouse brain. Arch Neurol 9:414-423, 1963 13. Baez S: Recording of microvascular dimensions with an im-
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14.
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age splitter television microscope. J Appl Physiol 2 1:299301, 1966 Baez S: A method for in-line measurement of lumen and wall of microscopic vessels in vivo. Microvasc Res 5:299-308, 1973 Rosenblum Wl: Effects of reduced hematocrit on erythrocyte velocity and fluorescein transit time in the cerebral microcirculation. Circulation Research 29:96-103, 1971 Greenberg S, Kadowitz PJ, Diecke FPJ, et ah Effect of prostaglandin F2O on arterial and venous contractility and "Ca uptake. Arch Int Pharmacodyn Therap 205:381-398, 1973 Greenberg S, Kadowitz PJ, Diecke FPJ, et ah Effect of prostaglandin F2a on responses of vascular smooth muscle to serotonin, angiotensin, and epinephrine. Arch Int Pharmacodyn Therap 206:5-18, 1973 Kapp J, Mahaley MS Jr, Odom GL: Cerebral arterial spasm. Part 2: Experimental evaluation of mechanical and humoral factors in pathogenesis. J Neurosurg 29:339-349, 1968 Kapp J, Mahaley MS Jr, Odom GL: Cerebral arterial spasm. Part 3: Partial purification and characterization of a
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spasmogenic substance in feline platelets. J Neurosurg 29:350-356, 1968 Wilkins RH, Wilkins GK, Gunnells JC, et ah Experimental studies of intracranial arterial spasm using aortic strip assays. J Neurosurg 27:490-500, 1967 Rosenblum Wh Effect of hypotension on sensitivity of minute cerebral vessels. Arch Neurol 23:266-270, 1970 Rosenblum Wh A possible role for mast cells in controlling the diameter of arterioles on the surface of the brain. Brain Res 49:75-82, 1973 Rosenblum Wh Contractile response of pial arterioles to norepinephrine. Effects in the mouse. Arch Neurol 31:197199, 1974 Rosenblum Wl, Donnenfeld H, Aleu F: Effects of increased blood pressure on cerebral vessels in mice. Arch Neurol 14:631-643, 1965 Welch KMA, Meyer JS, Teraura T, et ah Ischemic anoxia and cerebral serotonin levels. J Neurol Sci 16:85-92, 1972 Meyer JS, Stoica E, Pascu I, et ah Catecholamine concentrations in CSF and plasma of patients with cerebral infarction and haemorrhage. Brain 96:277-288, 1973
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Constriction of Pial Arterioles Produced by Prostaglandin F2α WILLIAM I. ROSENBLUM Stroke. 1975;6:293-297 doi: 10.1161/01.STR.6.3.293 Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1975 American Heart Association, Inc. All rights reserved. Print ISSN: 0039-2499. Online ISSN: 1524-4628
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• Prostaglandin F 2a constricted pial arterioles when locally applied to the cerebral surface. Norepinephrine and serotonin each elicited similar contractile effects. The constriction produced by F 2o in combination with either biogenic amine was greater than the constriction elicited by F2a or amine acting alone. The effect of one agent on the other was additive rather than potentiating. Since F2o, norepinephrine and serotonin are all naturally occurring agents, it is possible that their combined effect is important under pathological circumstances and this combined effect should not be overlooked in the search for single spasmogens of great potency. Before ascribing a pathologically important effect to F 2a , either alone or in combination, evidence is required showing that doses effective in experiments are similar to the concentrations occurring during disease states and/or that vessels may become hypersensitive to F 2a during such states.
Additional Key Words norepinephrine
• Prostaglandins (PG) are a group of 20-carbon, unsaturated lipid acids with a wide variety of possible biological actions, some of which are just beginning to be explored.1"3 They have been found to produce constriction or relaxation of smooth muscle, including vascular smooth muscle, in vitro and in vivo. The direction of response is dependent upon the type of PG and the test object. Similarly, they have been reported to facilitate or to inhibit adrenergic responses, sometimes by acting on the release of norepinephrine and sometimes by altering the responsivity of the test organ. Prior to this writing, the author is aware of only four reports describing the effect of the PG, known as F2n, on cerebral blood vessels. The results of this small number of studies suggested that additional investigation was warranted. Prior reports indicated that F2ct constricts blood vessels somewhere in the cerebral vasculature. 46 Two dealt, at least partly, with surface arteries and arterioles. One of these showed that intracarotid injection caused constriction of pial arteries and arterioles under 200 n in diameter, 6 while the other 5 demonstrated that intracisternal F2Q produced constriction of large surface arteries. These recent investigations suggested that the action of F 2a might be relevant to the clinical problem of vasospasm, especially since prostaglandins, including F 2o , are present in brain and released by brain into the cerebrospinal fluid (CSF). 7 9 A relationship between PGs and clinical vasospasm also is suggested by the fact that platelets release PGs,10 and a platelet facProfessor and Chairman, Division of Neuropathology, Medical College of Virginia, Box 17, Health Sciences Center, Richmond, Virginia 23298. Supported by HL 12775 and the John A. Hartford Foundation.
microcirculation serotonin
cerebral blood vessels vasospasm
tor(s) has been implicated in the vasospasm associated with subarachnoid hemorrhage. The present investigation focuses attention on the response of smaller pial arterioles, after local application of F 2a to the cerebral (i.e., arachnoid) surface. Under such circumstances, the PG will arrive at cerebral surface vessels (pial vessels) after crossing the arachnoid and passing through the CSF. Transport through the CSF also represents a route by which they would arrive at the cerebral blood vessels after release from brain or from platelets in the subarachnoid space. After initiating our studies, an additional report appeared11 describing constriction of pial arterioles and small arteries in the cat following application of F 2o to the cerebral surface. The technique used therein more closely resembles our own than do those in any of the earlier studies.
Methods Male mice, Swiss strain (Dublin Farms), were used. These weighed 18 to 35 gm and were anesthetized with urethane. A tracheotomy and craniotomy were performed and the dura stripped from the craniotomy site. The brain surface with the exposed pial vessels covered only by the transparent arachnoid was then irrigated continuously with a mock cerebrospinal fluid (Elliott's solution). This solution has the following composition: Na + 134 mEq/1; C a + + 2.7 mEq/1; M g + + 2 . 4 mEq/1; Cl 137 mEq/1; SO", 2.4 mEq/1; HPO< 1.5 mEq/1, and dextrose 800 mg/1. In addition the solution contains initially 2.3 mEq/1 HCO3, and is acidic. The pH rises on standing or mild heating and is readjusted to physiological levels (between 7.3 and 7.4) by bubbling CO 2 through the solution. This solution was delivered to the brain surface at the rate of 2 ml per minute by a constant perfusion pump which caused the fluid to pass through a heating coil and hypodermic needle. The orifice of the needle was a few millimeters
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above the craniotomy site and solutions emanating from the needle orifice fell in a continuous series of drops to the edge of the craniotomy and spread in a thin film over the cerebral surface, from whence the fluid was led by a cotton wick. The temperature of the fluid at the craniotomy site was 37°C. The pH of the fluid was monitored repeatedly during the observation of each mouse in the study, by sampling drops from the needle orifice. The pH of more than 100 measurements was 7.34 ± 0.03 (M ± SD). A side arm just proximal to the heating coil permitted delivery of a 1 ml test solution as a bolus interrupting the normal irrigant. This bolus took 2 minutes ± 30 seconds to reach the cerebral surface, at which time its temperature was the same as that of the normal irrigant. The pH of the bolus and that of the irrigant were within 0.05 pH units of each other. The bolus cleared the brain surface in 30 seconds, after which the regular irrigant was once again flowing over the brain. Solutions of 5% BaCl2 were used to test the reactivity of the pial arterioles before and after each experiment.12 All animals entering the study responded to the barium ion with an arteriolar constriction both before and after the application of F2(t. The prostaglandin F2Q used in this study was the tromethamine (THAM) salt, with a molecular weight of 475.6. The free acid has a molecular weight of 354.5. THAM, when used by itself over a dose range exceeding that applied in the present investigation (10" 5 -10 3 M), failed to alter the diameter of the pial arterioles. Norepinephrine (NOR) was used as the bitartrate and serotonin (5HT) as the creatinine sulfate. Fresh solutions of all agents were made each day in the Elliott solution and the pH adjusted to between 7.3 and 7.4 by adjusting the CO 2 in the solution. Preliminary studies with F2f, alone or combined with NOR showed no change in blood pressure during the local application of these agents to the pial surface and for five minutes thereafter. Thus during the 30-second period of application insignificant amounts of drug were absorbed into the blood stream. The internal diameter of the pial arterioles was measured by using a microscope, image splitter and TV monitor as described by Baez.13- " The internal diameter of the vessels was represented by a dark column of red cells and thus was underestimated by approximately 4 jt.15 The diameter of the vessels was monitored for five minutes prior to the time the test solution hit the brain, and continued to be monitored for eight minutes thereafter. Stability of baseline diameter prior to application of test solution was required and animals not showing such stability were discarded. If a change in diameter occurred within the 60 seconds following arrival of test solution at the cerebral surface, the change was counted as a response. In practice, over
90% of the responses occurred during the first 30 seconds. The point of maximal change in diameter was used to quantify the response. In these experiments the limiting factor in measuring diameter was the reproducibility with which successive measurements of the same vessel could be made. This depends upon the observer's ability to exactly juxtapose sheared images on the TV monitor and this, in turn, depends upon the clarity of the TV image and the contrast between vessel and background. In our system, which utilizes light reflected from the object being observed12'15 rather than transmitted light, we have tested our ability to make reproducible successive measurements of the distance between lines on a stage micrometer, a fixed distance demarcated by highly contrasted lines. These measurements never varied by more than 2 ju> and the reproducibility was independent of the distance between the lines over a range of 20 to 60 n. We took this 2 n discrepancy between successive measurements of a fixed object as the maximal "random" error of the method of measurement in our studies and arbitrarily discarded all changes smaller than 2 n from consideration as true changes. In other words changes of less than 2 n could not be distinguished from "noise." This change would equal approximately 4% of the mean resting diameter of arterioles in this study. Thus there will be a tendency to underestimate the frequency and magnitude of response to the vasoactive agents, especially at low dose levels.
Results EFFECTS OF SINGLE DOSES OF Fa,, ALONE
F 2a constricted pial arterioles as indicated in table 1. No relationship to dose could be seen until the lowest dose in the series, 10 ng per milliliter, where a marked decrement in response was observed. This decrement, however, was due solely to the failure of six of the ten animals to display any recognizable response, thus being counted as 0% constriction. In all other groups 70% to 100% of the animals responded to F 2a - Thus, in each group of ten mice, seven responded to 250 ng per milliliter, eight to 100 ng per milliliter, seven to 50 ng per milliliter, and all ten to 25 ng per milliliter. It is probable that the incidence of response was even higher, since small responses equal to less than 5% of resting diameter would have been dismissed as noise due to either spontaneous change in diameter or observer error in resetting the image splitter to the same null position between successive measurements.
TABLE 1
Effect of F, a Alone Dose F, a (fig/ml)* Number of experiments! Original diameter^ Percent constriction§
250 10 49 ± 15 11 ± 11
100 16 47 ± 12 14 ± 9
50 10 54 => = 13 12 ±9= 9
25 10 46 * 5 20 ± 7
10 10
45 ± 16 5 ± 10
*Administered as a 1 ml bolus over a 30-second period. fEquals number of mice. Only one dose per mouse per experiment. IMicrons =*= standard deviation. §Percent reduction from baseline diameter (mean =±= SD). 294
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PROSTAGLANDIN AND PIAL ARTERIOLES DOSE RESPONSE RELATIONSHIP TO F2(,
As indicated above, groups of animals failed to display a relationship between the average response of the group and the dose used in that group. We believe this was due to the great variability in the responses of the mice within each group. However, if successively larger doses were applied to the same animal, allowing the arteriole to recover from one dose before applying the next, greater constrictions were observed in the individual mouse as higher doses were applied to the pial surface of that mouse. This is shown in figure 1. The figure displays the results in five consecutive animals whose vascular reactivity was unaltered during the course of the experiment. The latter point was checked by comparing the response to BaCl2 before and after the series of F2«. In each animal the two responses to BaCl2 were within 10% of each other. INTERACTION OF Fto WITH SEROTONIN OR NOREPINEPHRINE
In each experiment an effective dose of amine was
EFFECT OF INCREASING DOSES OF F O O C
determined. This dose of amine was given alone, as was a dose of F 2a , and the response to each was measured. In addition, the same doses were combined and the response to the combination was measured. The order in which these drugs were given was varied, so that in some cases amine was given first, and in others the prostaglandin or the combination. The results were the same, irrespective of the order in which these agents were applied. The responses to F 2a were increased if 5HT or NOR were present at the same time. The response to the combination of drugs was equal to the sum of the responses to each of the components in the combination. The data are summarized in table 2, which displays the average response to the amine alone, to the F2a alone, and to the combination. In these experiments, the dose of NOR was either 0.5 or 5 ng per milliliter and the dose of 5HT was either 5, 10 or 20 jug per milliliter. The dose of F2Q was either 5, 25, or 50 ng per milliliter. The reader must remember that in each experiment the doses of amine and F 2o used singly were the same as those used in combination. The additive effect of NOR plus F 2Q or of 5HT plus F 2a was observed irrespective of the doses selected.
Discussion Our data clearly demonstrate constriction of pial arterioles after local application of F 2a . The results confirm the four previous reports7"9' u of which we are aware. The effective doses in our study are remarkably similar to those doses locally applied to the brain surface of the cat by Welch et al.11 in their study of the effect of F 2a on pial vessels. The latter is the only published study, of which we are aware, in which F 2Q was directly applied to the external surface of the brain and the pial vessels. Although we have not used duration of response as a parameter in this particular study, the response peak occurred between ten seconds and one minute following application of the drug, and thus the time course of the response was also similar to the data reported by Welch et al.11 These investigators did not report dose-response data, nor did they concern themselves with the interaction of F 2a and other vasoconstrictors. Our data show a simple, additive effect between TABLE 2
Interaction of F2a With Vasoactive Amines % Constriction*
80
FIGURE 1
Dose-response relationships are plotted for each of five mice. A single arteriole was monitored in each mouse, and the smallest diameter reached during the response was compared with the resting diameter. The decrement in diameter was expressed as a percent of the resting diameter.
Norepinephrine Prostaglandin F2a Combination Serotonin Prostaglandin Fna Combination
21 ± 8 22 ± 7 41 ± 14 13 ± 7 32 ± 10
N = 6f N = 6
42 ± 11
*Mean =•= standard deviation. Constriction as a percent of resting diameter. fN = number of experiments. 295
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ROSENBLUM
the response to F2a and the response to NOR or 5HT. These results contrast with those reported for isolated extracerebral vessels16'" where F2a was shown to potentiate the responses to 5HT and other contractile stimuli rather than produce a simple additive response. However, in the latter experiments much lower concentrations of F2Q were used, concentrations which themselves had no effect on vascular tone, and of course cerebral vessels were not tested. Our own results may be of some interest, since they suggest that marked constriction of cerebral arterioles may follow contact with a combination of agents, any one of which might produce only modest constriction if acting alone. Thus the search for a single, elusive "spasmogen"18"20 to account for clinically important vasospasm may be missing an important point, namely the additive effect of several agents released by platelets and brain tissue into the CSF and subarachnoid space. One must make further mention of the arteriolar constriction induced by either NOR or 5HT in these experiments. In earlier publications by this author the contractile response of pial arterioles to NOR was described as unreliable12 or nonexistent21 and negative data also were reported with respect to a contractile effect of 5HT.22 More recently, we have reported a reliable, dose-related constriction elicited by NOR.23 This is confirmed in the present experiments, and a constriction induced by 5HT is also described. Several differences in methodology have been introduced since the earlier negative studies. These include better control of pH and temperature; a shift from pentobarbital to urethane anesthesia; avoidance of applying to the same preparation multiple doses of vasoactive drug in maximal strength; and the use of the TV microscope and image splitter to provide reliable, sensitive measurements of diameter, and a written record of diameter changes during the experiment. We cannot say which of these factors may have contributed to the change in our results. We have tried pentobarbital anesthesia in conjunction with the new recording system and we continue to get constriction like that observed with urethane. Thus far, the reliability of the recording system seems extremely important, since in the past small transient constrictions induced by single applications of NOR or 5HT might have been overlooked or dismissed as illusory when viewed by peering directly down the microscope aided only by an ocular micrometer. However, extremely small changes were consistently recorded with the more primitive technique when other vasoactive drugs were used.24(lable"
Whatever the explanation for our previous inconsistent or negative data with biogenic amines, the present studies have been markedly consistent, provide evidence not only for a contractile effect of these agents but also for F2Q, and indicate an additive effect of the latter with either NOR or 5HT. Unfortunately, in order to solidly implicate F2a as a contractile
stimulus of clinical importance, or for that matter to implicate NOR or 5HT, one must show that their concentrations in these and other experiments are comparable to that occurring in disease states. This is not the case, and in the present study, as well as the studies of other workers, each of these vasoactive agents is effective only in concentrations greater than those known to occur even during pathological conditions.25- 26 However, the concentration of these materials during pathological states is still relatively unexplored and for PG is virtually unknown with respect to the central nervous system and cerebrospinal fluid. Moreover, the sensitivity of pial vessels may be enhanced during periods of microcirculatory derangement.2123 Thus it will be important to test responses to F2Q alone and in the presence of other vasoactive agents, when such derangements are present, in order to see whether marked constriction can be elicited by doses lower than those used here.
Acknowledgments Ms. Mary Allgood and Mr. Guy Nelson provided invaluable technical assistance.
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Constriction of Pial Arterioles Produced by Prostaglandin F2α WILLIAM I. ROSENBLUM Stroke. 1975;6:293-297 doi: 10.1161/01.STR.6.3.293 Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1975 American Heart Association, Inc. All rights reserved. Print ISSN: 0039-2499. Online ISSN: 1524-4628
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