239

J. Physiol. (1976), 259, pp. 239-249 With 6 text-figurem Printed in Great Britain

EVIDENCE THAT BRAIN PROSTAGLANDIN SYNTHESIS IS NOT ESSENTIAL IN FEVER

BY W. I. CRANSTON, G. W. DUFF, R. F. HELLON, D. MITCHELL* AND YVONNE TOWNSEND From the Department of Medicine, St Thomuas's Hospital Medical School, London SEl 7EH, and the National Institute for Medical Research, Mill Hill, London NW7 1AA

(Received 3 February 1976) SUMMARY

1. We have tested the hypothesis that a fever caused by pyrogen de-

pends upon the synthesis of prostaglandin E in the brain and that the prostaglandin in turn acts on the hypothalamus to produce fever. 2. In rabbits, fever was produced by the injection of leucocyte pyrogen in a lateral cerebral ventricle. The latency, rate of rise and magnitude of the fever was unaffected by the simultaneous intraventricular injection of two prostaglandin antagonists, SC 19220 and HR 546. 3. Both antagonists effectively attenuated the fever caused by the intraventricular injection of prostaglandin E2. 4. This evidence is not consistent with the hypothesis that prostaglandin E is the principal mediator of fever. INTRODUCTION

Evidence has accumulated over the last few years which suggests that increased synthesis of an E prostaglandin (PGE) in or near the anterior hypothalamus may be an essential process in the production of fever by pyrogens. The evidence is of three types. First, micro-injections of nanogram doses of PGE into the cerebral ventricles or anterior hypothalamus cause an immediate rise in body temperature (Milton & Wendlandt, 1971; Feldberg & Saxena, 1971 a; Potts & East, 1972; Hales, Bennett, Baird & Fawcett, 1973; Crawshaw & Stitt, 1975). Secondly, there is an increased concentration of PGE in cerebrospinal fluid (c.s.f.) during fever induced by pyrogen (Feldberg & Gupta, 1973; Philipp-Dormston & Siegert, 1974a, b; Cranston, Hellon & Mitchell, 1975). Thirdly, antipyretic drugs, such as Present address: Department of Physiology, University of the Witwatersrand Medical School, Hospital Street, Johannesburg 2001, South Africa. *

W. I. CRANSTON AND OTHERS paracetamol and indomethacin, inhibit the synthesis of prostaglandins (Vane, 1971; Flower, 1974); these drugs attenuate a fever following the injection of pyrogen and at the same time reduce the level of PGE in c.s.f. (Feldberg, Gupta, Milton & Wendlandt, 1973; Dey, Feldberg, Gupta, Milton & Wendlandt, 1974). We have recently questioned the validity of the third type of evidence (Cranston et al. 1975). Administration of an appropriate dose of salicylate to rabbits prevented the release of PGE in c.s.f. during a pyrogen fever, but had no effect on the fever. In the present experiments we have tested the effect of two prostaglandin antagonists, a dibenzoxazepine derivative, SC 19220 (Sanner, 1969; Bennett & Posner, 1971) and a substituted prostaglandin A, HR 546 (Scholkens & Babej, 1975), on the elevations of body temperature in rabbits caused by leucocyte pyrogen (LP) and PGE. If the increased PGE synthesis following pyrogen is an essential process in fever, and if the antagonists inhibit the effects of PGE in brain, then both elevations of temperature should be diminished by the antagonists. This hypothesis has been tested in the present experiments. Some of the results have been communicated to the Physiological Society (Cranston, Duff, Hellon & Mitchell, 1976). 240

METHODS

Rabbits of either sex weighing between 2-3 and 3-2 kg were used. At a preliminary operation under general anaesthesia a stainless-steel plate was screwed to the skull (Monnier & Gangloff, 1961). At least 1 week then elapsed before any experiments were made on the rabbits. In all the experiments room temperature was maintained between 21 and 230 C. The rabbits were restrained in conventional stocks. Rectal temperature, measured by an indwelling thermistor, was recorded automatically at 5 min intervals. Using techniques described previously (Cranston, Luff, Rawlins & Wright, 1971) a guide tube was screwed to the skull plate. A sterilized stainless-steel cannula (0-56 nun o.d.) was lowered into a lateral cerebral ventricle. The cannula was connected to a 1 ml. syringe by a finee nylon tube and 100 #1. artificial c.s.f. (Cameron & Semple 1968) was injected intraventricularly. One hour was allowed to elapse before further injections were made to ensure that the system was not contaminated by pyrogens. If no contamination was evident, one of fourteen types of intraventricular injection was administered, and the rabbit's response monitored for a further 3 hr. The composition of the fourteen types of injection are set out in Table 1. To make an injection, the cannula and nylon tube were first removed from the rabbit. A Hamilton syringe (100 el.), partially loaded with artificial c.s.f., was attached to the end of the nylon tube and the composite injection was slowly drawn in through the cannula. About 2 #1. air was sucked in between different solutions. Each injection except no. 8 was followed by 30 #l. artificial c.s.f. The doses of LP and PGE2 were chosen so as to produce submaximal fevers of approximately equal magnitude. The dibenzoxazepine antagonist, 1-acetyl-2-(8-chloro-10,11-dihydrobenz (b, f) (1, 4) oxazepine-10-carbonyl) hydrazine (SC 19220) was dissolved in dimethyl

FEVER AND PROSTAGLANDIN SYNTHESIS

241

sulphoxide. The substituted prostaglandin A antagonist, 8-ethoxycarbonyl-10,11dihydro-A-prostaglandin (HR 546) was dissolved in ethanol. Prostaglandin E2, as the sodium salt, was dissolved in isotonic sodium chloride solution. TABLE 1. Summary of intraventricular injections used Mean rectal temperature

1 2

3 4

5 6 7 8 9 10

11 12 13 14

Treatment 15 Emole SC 19220 in 20 dll. DMSO 15 molee SC19220 in 20,t1. DMSO+ 2-5 n-mole PGE2 in 15 ,d. isotonic saline 15 Emole SC19220 in 20 4a1. DMSO + 15 dl. saline containing LP 20 10. DMSO + 2-5 n-mole PGE2 in 15 dl. isotonic saline 20 jul. DMSO + 15 1d. saline containing LP 20,ul. DMSO 15 molee SC 19220 in 20 1d. DMSO, 20 min pause, 2-5 n-mole PGE2 in 15 1l. isotonic saline into opposite ventricle 70 #1. c.s.f. 20 EM. ethanol 20 il. ethanol + 2-5 n-mole PGE2 in 30 el. isotonic saline 440 n-mole HR 546 in 20 #1. ethanol + 2-5 n-mole PGE2 in 30 #1. isotonic saline 440 n-mole HR 546 in 20 ,d. ethanol 20 #41. ethanol + 15 #1. saline containing LP 440 n-mole HR 546 in 20 Id. ethanol + 15 #1. saline containing LP

No. of animals 6 10

(0C±S.E.)

at

time of

injection 38-89 ± 0-24 39*44+0*73

6

39-11+0-54

9

38-99 + 0 53

6 5 8

39.45 + 0*39

6 6 9

39-19 ± 0-45 38-48 + 0-08 38-66 + 0-24

9

38-79 + 0-20

6 8 8

38-57+0-20 38-85 + 0-14 38-99 + 0-17

39 34 + 0-27 39.39 ± 0-62

Abbreviations: DMSO, dimethyl sulphoxide; LP, leucocyte pyrogen. See text for details of SC 19220 and HR 546. In all treatments, except no. 8, the drug injection was washed in with 30-4()0ul. c.s.f. Because SC 19220 has a very low solubility in aqueous media and in ethanol, dimethyl sulphoxide was used as its solvent. When the solution of PGE in dimethyl sulphoxide was added to rabbit artificial c.s.f. in vitro, precipitation was observed. Precipitation was also found po8t mortem in the lateral ventricle of animals injected with this solution; in animals killed up to 4 weeks after injection, ipsilateral ventricular dilatation was sometimes evident, suggesting that the precipitate might have occluded the foramen of Monro. In no animal was there evidence of any occlusion lower down the c.s.f. pathway or of the opposite foremen of Monro. Because of the persistence of precipitated SC 19220, experiments could not be carried out to a balanced randomized design. Injections of SC 19220 were always given as the last injection to an animal and no animal received more than one such injection. The possibility was considered that a precipitate of SC 19220 might have prevented access of PGE2 or LP to the third ventricle, but it seemed unlikely that this would

242 W. I. CRANSTON AND OTHERS have affected one substance more than another. To exclude this possibility, a further series of experiments were carried out (treatment 7, Table 1). Here SC 19220 was injected into one lateral ventricle and after a delay of 20 min PGE2 was injected into the opposite ventricle. Leucocyte pyrogen was made by incubating rabbit whole blood with Proteus endotoxin (E pyrogen, Organon Laboratories) at a concentration of 3 ng ml.-' for 2 hr at 370 C. The cells were separated by centrifuging at 2000 g for 20 min, washed with sterile saline, suspended in saline of volume equal to the original volume of plasma, and incubated for a further 18 hr. The supernatant was separated by centrifuging as before, and stored at 40 C until used. RESULTS

None of the injections was followed by any change in the animal's behaviour or appearance. Mean rectal temperatures for each treatment group at the time of injection are given in Table 1.

Experiments with SC 19220 The mean changes in rectal temperature following the intraventricular injection of dimethyl sulphoxide (DMSO) alone, SC 19220 dissolved in DMSO, and c.s.f. alone (treatments 1, 6 and 8, Table 1) are plotted in Fig. 1. In each group of rabbits there was a slow rise in temperature amounting to about 0.50 C over the 3 hr of observation. Differences between the treatments, assessed with t tests, were not significant. The SC I| X Ie | Ie |

15

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Fig. 1. Mean changes in rectal temperature before and after time of intraventricular injection at arrow. The three curves show effects of artificial c.s.f. (E, n = 6) dimethyl sulphoxide (DMSO) (0, n = 5) and SC 19220 (@, n = 6) in dimethyl sulphoxide. For details of injections, see Table 1. The bars indicate S.E.; standard errors of the mean responses to DMSO were similar in magnitude and were therefore omitted for clarity.

243 FE VER AND PROSTAGLANDIN SYNTHESIS 19220 itself therefore had no effect on the temperatures of the afebrile animals. In the results which follow, the values for the mean temperature change at each 10 min interval after injections of the appropriate vehicles have been subtracted from individual changes following the injection of test substances; for substances dissolved in saline the corresponding response to c.s.f. was subtracted. This subtraction procedure removed the complicating factor of a slow non-specific temperature rise (Fig. 1) in the analysis of the effects of LP, PGE and antagonist. 15

I

O.00.

E

0-05 U0u 0-05 C

-30

0

30

60

90

120

150

180

Time (min)

Fig. 2. Changes in rectal temperature (as in Fig. 1) after intraventricular injections of PGE2 (0, n = 9) and PGE2 with SC 19220 (O, n = 10). For details of injections, see Table 1. Mean temperature changes following injections of the appropriate vehicles alone have been subtracted in both cases.

Fig. 2 shows the effects on rectal temperature of intraventricular injections of PGE2, with and without the simultaneous injection of SC 19220 (treatments 2 and 4, Table 1). PGE2 alone caused a steady rise of temperature, which reached a peak 50 min after injection and then fell slowly over the following 2 hr. There was no such rise in the presence of SC 19220. The differences between the curves were significant at the 5 % level or better at 20, 30, 40 and 50 min after injection. By contrast, the fever caused by leucocyte pyrogen was quite unchanged by SC 19220 (treatments 3 and 5, Table 1). The mean changes in rectal temperature, shown in Fig. 3, are almost identical. Only at 110 min is there a statistical difference between the two sets of data, and in this instance the mean temperature following LP with SC 19220 was greater than that following LP alone. Comparison of Figs. 2 and 3 shows that the onset of pyrexia was much slower with LP than with PGE2. Thus there was the possibility that the

244 WV. 1. CRANSTON AND OTHERS antagonist might no longer have been active at the time of onset of a fever following LP injection. To test this possibility an additional series of experiments was conducted (Table 1, Treatment 7). SC 19220 was injected intraventricularly 20 min before injection of PGE2. The response to this treatment is compared in Fig. 4 with the response to the same two agents given simultaneously. There was no significant difference between the responses; if anything, the temperature rise was smaller if the injection of SC 19220 was given 20 min before the prostaglandin. The failure of SC 19220 to affect LP fever therefore cannot be attributed to inactivation of the antagonist. U

05

0

.0

U

_'0-5

60 90 120 150 180 Time (min) Fig. 3. Changes in rectal temperature (as in Figs. 1 and 2) after intraventricular injections of LP (O. n = 6) and LP with SC 19220 (@, n = 6). For details of injections, see Table 1. -30

10. _

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120

180

Fig. 4. Changes in rectal temperature (as in Figs. 1 and 2) after intraventricular injection of PGE2 with SC 19220 given together into one ventricle ( *, n = 10) and SC 19220 given into one ventricle at minus 20 min followed by PGE2 into the opposite ventricle at arrow (U, n = 9). For details of injections, see Table 1.

FEVER AND PROSTAGLANDIN SYNTHESIS II

liii

III

III

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liii

245

III

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'4

co

05

41

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-60

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60 30 Time (min)

0

180

150

120

Fig. 5. Changes in rectal temperature (as in Figs. 1 and 2) after intraventricular injections of PGE2 (-, n = 9) and PGE2 with HR 546 (O. n = 9). For details of injections, see Table 1.

I

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1111

1111

1111

111

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180

Fig. 6. Changes in rectal temperature (as in Figs. 1 and 2) after intraventricular injections of LP (@, n = 8) and LP with HR 546 (0, n = 8). For details of injections, see Table 1.

246

W. I. CRANSTON AND OTHERS

Experiment with Hr 546 Intraventricular injections of HR 546 dissolved in ethanol and of ethanol alone (treatments 9 and 12, Table 1) were followed by a slow rise in rectal temperature similar to the corresponding control experiments with SC 19220 (Fig. 1). As before, the mean values for the vehicle experiments have been subtracted from the individual changes following injection of each test substance. The hyperthermic effect of PGE2 was considerably attenuated by the presence of HR 546 (Fig. 5) during the first hour following injection. The mean values were significantly different at the 5 % level, or better at 10, 20, 30, 40 and 50 min after injection. HR 546 had no effect on the fever caused by LP, as is shown in Fig. 6. There was no statistical difference between the two sets of mean values. DISCUSSION

Previous attempts have been made to use SC 19220 as an antagonist against pyrogen fever. Sanner (1974) briefly reported experiments in which intraperitoneal injections of SC 19220 were found not to diminish fever in rabbits. In cats, intravenous injection of SC 19220 alone caused marked hypothermia, vomiting and numerous other side-effects (Clark & Cumby, 1975). During the hypothermia neither pyrogen nor PGE1 caused any significant fever. Because the systemic administration of SC 19220 gave hypothermia coupled with many behavioural side effects, we used the intraventricular route; no untoward responses were seen following injection of SC 19220 in this way. The two prostaglandin antagonists we have used differ both in structure and mode of action. SC 19220, a dibenzoxazepine derivative, is a selective antagonist against PGE2 and PGF2. when tested on guinea-pig and rat smooth muscle preparations (Bennett & Posner, 1971). The antagonism is competitive. SC 19220 does not act by direct chemical action on PGE2, since it does not block the contractile effect of PGE2 on human smooth muscle. In contrast, HR 546, a substituted prostaglandin A compound, has a non-competitive type of action and is not a specific antagonist for prostaglandins; the contractile actions of histamine and carbachol chloride are also blocked (Sch6lkens & Babej, 1975). Despite their pharmacological dissimilarities, both antagonists had similar effects in the present experiments. A submaximal fever caused by the intraventricular injection of LP was quite unchanged by the intraventricular injection of either of the antagonists. There was no difference in latency of onset, in rate of temperature rise or in peak temperature.

FE VER AND PROSTAGLANDIN SYNTHESIS 247 A fever of the same magnitude caused by PGE2 was markedly reduced in the presence of the antagonists. SC 19220 abolished the rise in temperature during the first hour following injection of PGE2. HR 546 did not have a total blocking effect, but it did significantly reduce the hyperthermic action of PGE2. The simplest interpretation of these findings is that any brain prostaglandin synthesized during pyrogen fever does not play an essential role in the genesis of the fever. Before considering the implications of this conclusion we must first discuss other possible interpretations. It could be argued that the antagonists were not acting by blocking the action of synthesized prostaglandin, but by selectively preventing the passage of PGE2, but not LP, through the ependyma of the third ventricle into the pre-optic area. The pre-optic area is the sole site of action for the hyperthermic effect of exogenous PG (Feldberg & Saxena, 1971b; Stitt, 1973) and the main site of action of pyrogen (Cooper, Cranston & Honour, 1967; Jackson, 1967; Repin & Kratskin, 1967; Rosendorff & Mooney, 1971). It seems unlikely that both antagonists with their disparate structures, would act in such a way. Another explanation could be based on the fact that SC 19220 precipitates from its solvent, DMSO, when mixed with an aqueous medium such as c.s.f. As indicated in methods, a deposit of SC 19220 was observed in the injected lateral ventricle for a considerable time after injection. The deposit could have mechanically blocked access from lateral to third ventricles. If so, the blockage would have to be selective for PGE2 but not LP. Also, SC 19220 was equally effective in blocking PGE2 fever when injected into the opposite lateral ventricle. A final possibility which might be entertained is that LP does produce fever through the mediation of endogenously synthesized PGE. If this were the case, one would have to postulate that in our experiments such PGE acts on receptors which are not accessible to antagonists given intraventricularly i.e., the receptors are different from those activated when PGE itself is injected into the ventricles. While this possibility. cannot be rigorously excluded, it seems to us to be remote. We thus return to the most ready interpretation of our findings, namely that the production of fever is not dependent on any increased synthesis of PGE which may have been demonstrated. Although the precise mode of action of pyrogen at the cellular level remains unclear, it can now be assumed that pyrogen molecules must either act directly on elements in the pre-optic area or through some mediator which is not a prostaglandin. It also follows that although antipyretic drugs have the property of inhibiting prostaglandin synthesis, this is unlikely to be their mode of action in fever.

248

W. I. CRANSTON AND OTHERS

We are grateful to G. D. Searle and Co. Ltd for supplies of SC 19220; to Hoechst AG (Frankfurt) for supplies of HR 546; to The Upjohn Company for supplies of PGE2; and to the Pharmacy, St Thomas's Hospital for making artificial c.s.f. solution. REFERENCES BENNETT, A. & POSNER, J. (1971). Studies on prostaglandin antagonists. Br. J. Pharmac. 42, 584-594. CAMERON, I. R. & SEMPLE, S. J. G. (1968). The central respiratory stimulant action of salicylates. Clin. Sci. 35, 391-401. CLARx, W. G. & CUmBY, H. R. (1975). Effects of prostaglandin antagonist SC 19220 on body temperature and on hyperthermic responses to prostaglandin E1 and leukocytic pyrogen in the cat. Pros8tglandin8 9, 361-368. COOPER, K. E., CRANSTON, W. I. & HONOUR, A. J. (1967). Observations on the site and mode of action of pyrogens in the rabbit brain. J. Physiol. 191, 325-337. CRANSTON, W. I., DUFF, G. W., HELLON, R. F. & MITCHELL, D. (1976). Effect of a prostaglandin antagonist on the pyrexias caused by PGE2 and leucocyte pyrogen in rabbits. J. Physiol. 256, 120-121 P. CRANSTON, W.I., HELLON, R. F. & MITCHELL, D. (1975). A dissociation between fever and prostaglandin concentration in cerebrospinal fluid. J. Phy8iol. 253, 583-592. CRANSTON, W. I., LUFF, R. H., RAWLINS, M. D. & WRIGHT, V. A. (1971). Influence of the duration of experimental fever on salicylate antipyresis in the rabbit. Br. J. Pharmac. 41,344-351. CRAWsHAW, L. I. & SnTT, J. T. (1975). Behavioural and autonomic induction of prostaglandin E1 fever in squirrel monkeys. J. Phy8iol. 244, 197-206. DEY, P. K., FELDBERG, W., GUPTA, K. P., MILTON, A. S. & WENDLANDT, S. (1974). Further studies on the role of prostaglandin in fever. J. Phy8iOl. 241, 629-646. FELDBERG, W. & GIJPTA, K. P. (1973). Pyrogen fever and prostaglandin activity in cerebrospinal fluid. J. Physiol. 228, 41-53. FELDBERG, W., GUPTA, K. P., MILTON, A. S. & WENDLANDT, S. (1973). Effect of pyrogen and antipyretics on prostaglandin activity in cisternal c.s.f. of unanaesthetized cats. J. Phy8iol. 234, 279-303. FELDBERG, W. & SAXENA, P. N. (1971a). Fever produced by prostaglandin E1. J. Phyaiol. 217, 547-556. FELDBERG, W. & SAXENA, P. N. (1971b). Further studies on prostaglandin E1 fever in cats. J. Phy8iol. 219, 739-745. FLOWER, R. J. (1974). Drugs which inhibit prostaglandin biosynthesis. Pharmacy. Rev. 46, 33-67. HALES, J. R. S., BENNETT, J. W., BAIRD, J. A. & FAWCETT, A. A. (1973). Thermoregulatory effects of prostaglandins E1, E2, F,. and F2. in the sheep. Pfluger8 Arch. ge8. Physiol. 339, 125-133. JACKSON, D. L. (1967). A hypothalamic region responsive to localized injection of pyrogens. J. Neurophy8iol. 30, 586-602. MILTON, A. S. & WENDLANDT, S. (1971). Effect on body temperature of prostaglandins of the A, E and F series on injection into the third ventricle of unanaesthetized cats and rabbits. J. Physiol. 218, 325-336. MONNIER, M. & GANGLOFF, H. (1961). Atka8 for Stereotaxic Brain Research on the Consciou8 Rabbit. Amsterdam: Elsevier. PHIILIPP-DORMSTON, W. K. & SIEGERT, R. (1974a). Identification of prostaglandin E by radioimmunoassay in the cerebrospinal fluid during endotoxin fever. Naturunimsenmchaften 61, 134-135.

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PHILIPP-DORMSTON, W. K. & SIEGERT, R. (1974b). Prostaglandins of the E and F series in rabbit cerebrospinal fluid during fever induced by Newcastle Disease Virus, E. coli - endotoxin, or endogenous pyrogen. Med. Microbiol. Immunol. 159, 279-284. POTTS, WV. J. & EAST, P. F. (1972). Effects of prostaglandin E2 on the body temperature of conscious rat and cat. Arche int. Pharmacodyn. Ther. 197, 31-36. REPIN, I. S. & KRATSKIN, I. L. (1967). On the analysis of hypothalamic mechanism in the pyretic reaction. Fiziol. Zh. SSSR. 53, 336-340. ROSENDORFF, C. & MOONEY, J. J. (1971). Central nervous system sites of action of a purified leucocyte pyrogen. Am. J. Physiol. 220, 597-603. SANNER, J. H. (1969). Antagonism of prostaglandin E2 by 1-acetyl-2-(8-chloro-10, 11-dihydrobenz (b, f) (1, 4) oxazepine-10-carbonyl) hydrazine (SC 19220). Arche int. Pharmacodyn. Ther. 180, 46-56. SANNER, J. H. (1974). Substances that inhibit the actions of prostaglandins. Arch8 intern. Med. 133, 133-146. SCHOLKENS, B. A. & BABEJ, M. (1975). Effect of prostaglandin antagonists on smooth muscle contraction. Naunyn-Schmiedeberge Arch. Pharmacol. 287, R44. STIrT, J. T. (1973). Prostaglandin E1 fever induced in rabbits. J. Physiol. 232, 163-179. VANE, J. R. (1971). Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature, New Biol. 231, 232-235.