J. Physiol. (1975), 253, pp. 583-592
With 4 text-figures Printed in Great Britain
A DISSOCIATION BETWEEN FEVER AND PROSTAGLANDIN CONCENTRATION IN CEREBROSPINAL FLUID
BY W. I. CRANSTON, R. F. HELLON AND D. MITCHELL From the Department of Medicine, St Thomas's Hospital Medical School, London SE1 7EH, and the National Institute for Medical Research, London NW7 1AA (Received 10 June 1975) SUMMARY
1. Sustained fever has been induced in conscious rabbits by i.v. injection and infusion of endogenous pyrogen. 2. Cerebrospinal fluid (c.s.f.) was sampled from the cisterna magna at hourly intervals. The concentration of prostaglandin increased in parallel with rectal temperature. The prostaglandin was identified as one of the E series. 3. When sodium salicylate (145 m-mole followed by a continuous infusion of 9 /tmole/min) was started 1 hr before endogenous pyrogen, the febrile response to the pyrogen was not significantly diminished but no rise of prostaglandin concentration was detected in c.s.f. 4. This dissociation between fever and prostaglandin concentration means that changes in cisternal prostaglandin concentration cannot be accepted as evidence that prostaglandin mediates the febrile response. INTRODUCTION
Fever results from the action on the brain, particularly the preoptic and anterior hypothalamic areas, of endogenous pyrogen, which is released by cells in response to endotoxins and many other agents (Atkins & Bodel, 1971; Hellon, 1974). There is a growing body of circumstantial evidence which implicates brain prostaglandin synthesis in the action of pyrogens. Prostaglandins of the E series (PGE) cause hyperthermia when introduced in small doses into the brain of many species (Milton & Wendlandt, 1971; Feldberg & Saxena, 1971a; Potts & East, 1972; Hales, Bennett, Baird & Fawcett, 1973; Crawshaw & Stitt, 1975). Their main site of action is indistinguishable from the main site of action of pyrogens (Feldberg & Saxena, 1971 b; Stitt, 1973). During fever produced by endotoxin (Feldberg & Gupta, 1973; Philipp-Dormston & Siegert, 1974a) or endogenous
W. 1. CRANSTON AND OTHERS pyrogen (Philipp-Dormston & Siegert, 1974b) there is an increased prostaglandin-like activity in cerebrospinal fluid (c.s.f.). The aspirin-like drugs, which are known to inhibit brain prostaglandin synthesis in vitro (Flower, 1974), not only reduce fever but also prevent the release of PGE into c.s.f. (Feldberg, Gupta, Milton & Wendlandt, 1973; Dey, Feldberg, Gupta, Milton & Wendlandt, 1974). If prostaglandin synthesis forms an essential link in the action of pyrogens on the brain, then it should not be possible to dissociate fever and brain prostaglandin synthesis in any circumstances. In the present experiments we tested the association between fever and prostaglandins in two ways. The first was to compare the time courses of body temperature elevation and prostaglandin release into c.s.f. We employed continuous infusions of endogenous pyrogen to achieve a steady-state fever (Cranston, Luff, Rawlins & Rosendorff, 1970). All previous work on the relation between prostaglandin release and fever has been concerned with fevers following single injections of pyrogen. The second was to examine the action of sodium salicylate which, though inhibiting prostaglandin synthesis (Flower 1974), is only weakly antipyretic in rabbits (Grundman, 1969; Cranston et al. 1970; van Miert, van Essen & Tromp, 1972), especially if administered early in the course of fever (Cranston, Luff, Rawlins & Wright, 1971). A brief account of the findings has appeared previously (Cranston, Hellon & Mitchell, 1975). METHODS
New Zealand White or Chinchilla rabbits of either sex, weighing 2-25-3-5 kg, were used. At least 7 days before an animal was used in an experiment, a stainless-steel guide tube was implanted with its tip just above the atlanto-occipital membrane over the cisterna magna (Feldberg et al. 1973). Animals were anaesthetized with intravenous alphaxolone and alphadolone (Althesin, Glaxo Laboratories Ltd) and strict aseptic precautions were observed. All the experiments were conducted in an air-conditioned room where the temperature was maintained between 21-23° C. The rabbits were unanaesthetiied but restrained in conventional stocks. Rectal temperature, measured by an indwelling thermistor, was automatically recorded every 5 min. A fine nylon tube was introduced into a marginal vein of the ear to allow the administration of drugs. Each experiment involved the administration of drugs for 41 hr. Each administration consisted of a rapid injection of a priming dose of the drug, followed by a sustained slow infusion maintained by motor-driven syringes. Three programmes of administration were employed (see Fig. 1). Six animals were subjected to programme A, which consisted of the intravenous administration of physiological saline (150 mmole/l. NaCl solution) for the full period. Eight animals were subjected to B, in which saline was administered as in A and after 1 hr the rabbits received an injection and infusion of homologous plasma containing endogenous pyrogen. Finally, eight animals were subjected to C, which consisted of the administration of a Na salicylate solution (80 m-mole/l.) at the same volumetric rate and for the same period as saline
FEVER AND PROSTAGLANDIN RELEASE
had been administered in A and B, together with pyrogen as in B. The time at which pyrogen was first administered (i.e. 1 hr after the beginning of each experiment) was called time zero. No experiments were done using salicylate alone, since Cranston et al. (1970) have already shown that similar infusions to those used here were without effect on the temperature of rabbits.
A [ Wm-mole
Saline ~~18 ,umole/min
[ 3 m-mole l l B
0 02 ml./min
Na salicylate 9 ,umole/min
11*5 m-mole 15rn-mole
2 ml./min 0[lPyrogen
3 ml. I
120 60 Time (min)
Fig. 1. Diagrams to illustrate the time courses of the three experimental programmes. The concentrations of the saline and salicylate solutions are given in the text. The method of preparation of endogenous pyrogen has been described previously
(Cranston et al. 1970). Rabbit whole blood was incubated with purified Proteus endotoxin ('E' pyrogen, Organon Laboratories) at a concentration of 3 ng/ml. for 18 hr at 370 C. The plasma was separated by centrifugation at 2000 g for 30 min and stored at 40 C until used. The same batch of plasma was used throughout the experiments. A sterile sharpened stainless-steel cannula was used for the collection of c.s.f. samples. At the start of each experiment it was lowered into the implanted guide tube until the membrane over the cisterna was pierced and c.s.f. flowed out. The cannula was then fixed in position, and serial samples drawn off through a siphon tube. Five samples were drawn off in each experiment: at -20 min, 30, 90, 160 and 200 min. The volume of each sample was 0-6 ml. The c.s.f. was assayed for prostaglandin-like activity on the rat fundus strip
W. I. CRANSTON AND OTHERS
preparation rendered insensitive to 5-hydroxytryptamine and rendered incapable of endogenous prostaglandin synthesis (Feldberg et al. 1973). Prostaglandin E2 was used as the standard. The method of measurement had a threshold of about 3 p-molefl. (1.1 ng/ml.). Any sample with activity below threshold was assigned the concentration 3 p-mole/l. In another experiment samples of c.s.f. were taken by the same method from three rabbits after administration of pyrogen from the same batch. These samples were pooled, and aliquots were extracted and separated using thin layer chromatography to identify the prostaglandin-like constituent (Feldberg et al. 1973). Aliquots were also subjected to radio-immunoassay for PGE (Dozois & Thompson, 1974). (These assays were made by Susan Cammock of the Department of Pharmacology, University of Aberdeen.) RESULTS
All rectal temperatures were expressed as changes from the temperature at zero time. The actual temperatures of the three groups of rabbits at time zero are shown in Table 1, and were not significantly different. TABLE 1. Rectal temperatures of rabbits at time zero (see Fig. 1)
Rectal temperature (0 C) Programme A B C
No. of animals 6 8 8
39.33 39-07 39-20
s.E. of mean 0-20 0-20 0-13
Range 38-7-40-2 38-4-39-9
Fig. 2 shows the mean change of rectal temperature, at 10 min intervals from time zero, of the six rabbits given intravenous saline only (programme A). There was a slight rise in rectal temperature (about 0.20 C) after time zero but at no stage was the rise statistically significant. Also shown in Fig. 2 are the levels of prostaglandin-like activity found in samples of c.s.f. taken during the same period. The mean activity was low at all times. In four of the six rabbits it was below the threshold of the assay at the first, second and third samples. Mean activity was slightly elevated in the fourth and fifth samples. Analysis of variance showed a significant time trend in the activity levels (F3; 20 = 4-6, P < 0-05), but the mean activity reached at the fifth sample was still only equivalent to 5-5 + 1-4 p-mole/1. (2.0 + 0-5 ng/ml.) PGE2. Thus the experimental procedure and the i.v. administration of saline was associated with a small and delayed rise in prostaglandin-like activity in the c.s.f., but no significant change in rectal temperature. The rectal temperature changes of the eight rabbits receiving saline and endogenous pyrogen (programme B) are shown in Fig. 3. After a latency of 10 min following the start of pyrogen administration, rectal temperature rose steadily for the next 40 min, reaching a plateau which
FEVER AND PROSTAGLANDIN RELEASE uI
I l IIIII I II1111I1I1
I . I I
I I I
120 Time (min)
Fig. 2. Responses of six animals to saline infusion starting at -60 min (programme A). Upper record: change in rectal temperature, Tre (mean + S.E. of mean) from time zero. Lower record: prostaglandin activity (mean + S.E. of mean) in cisternal c.s.f. IiI I II I I
I 1 1 1 1 1 1 1 1 1
0*5 OC Z 0.
I i I
c E ._
. . . .
2 0 .
120 Time (min)
Fig. 3. Responses of eight animals to saline infusion starting at -60 min and pyrogen infusion starting at 0 min (programme B). Records as in Fig. 2.
588 W. I. CRANSTON AND OTHERS persisted for the remainder of the infusion period. The mean change in rectal temperature was significantly different from that during saline infusion (t = 3-1, P < 0.01) at all times following the 10 min latent period. Fig. 3 also shows the mean levels of prostaglandin-like activity in c.s.f. before and during the pyrogen administration. The level before pyrogen administration was not significantly different from the level in the sample withdrawn at a similar stage during programme A. Samples withdrawn at all stages during the pyrogen administration showed elevated activity. Analysis of variance demonstrated no significant effect of time on the level of activity (F3;27 = 1.2); the activity therefore reached a I.111.1I1l
l lI*l l
l l l11l11l1
60 120 Time (min)
Fig. 4. Responses of eight animals to Na salicylate infusion starting at -60 mm and pyrogen infusion starting at 0 m2 (programme C). Records as in Fig. 2.
constant level 30 mmn after the start of pyrogen administration and maintained this level as long as the infusion continued. Because time had no effect on the activity level, values obtained during the pyrogen infusion could be pooled. The mean level was equivalent to the activity of 9'9 + 144 p-mole/l. (3.6 ± 0 5 ng/ml.) PGE2, which was highly significantly different from the equivalent mean level during saline infusion (t =3.7, P < 0.01). Fig. 4 shows the results from the group of rabbits receiving a continuous infusion of salicylate to which leucocyte pyrogen was added at zero time (programme C). A rise in rectal temperature occurred which was very
FEVER AND PROSTAGLANDIN RELEASE 589 similar to that in the rabbits not receiving salicylate. Direct comparison of the mean change in rectal temperature at each 10 min stage revealed a significant difference only at the 90th min and 150th min (t = 2-2, P < 0.05). Thus in the rabbits receiving salicylate, rectal temperature rose with the same latency and rate to a level not significantly different from that reached in the rabbits not receiving salicylate, and maintained a level not significantly different except for two isolated instances. In contrast, salicylate prevented completely the increase in c.s.f. prostaglandin levels. Analysis of variance once more demonstrated no effect of time on the prostaglandin-like activity during the pyrogen administration (F3;25 = 2 4). The mean activity of all samples taken during infusion was equivalent to that of 4-7 + 0 5 p-mole/l. (13 + 0-2 ng/ml.) PGE2. This value was significantly different from the comparable value in the rabbits not receiving salicylate (t = 4.5, P < 0-001) but not different from the comparable value in the rabbits receiving saline alone (t = 0.5). Thus in the group of rabbits to which salicylate was administered, pyrogen produced a fever not significantly different from that produced without salicylate, but without any increase of prostaglandin-like activity in c.s.f. One explanation for the failure to detect prostaglandin in the c.s.f. of rabbits receiving salicylate might be that salicylate in the c.s.f. samples interfered with the bio-assay. Salicylate concentrations in the c.s.f. were not measured but it is known from other experiments on rabbits with similar doses of salicylate (Rawlins, Luff & Cranston, 1973) that plasma concentrations reach about 1*3 m-mole/l. It is most unlikely that c.s.f. values would approach this level. When standard solutions of PGE2 were assayed on the rat fundus strip with and without the addition of salicylate to a 2 m-mole/l. concentration, there was no detectable difference in activity. Therefore the failure to detect prostaglandin in the c.s.f. of the rabbits receiving salicylate was not due to the action of salicylate on the rat fundus strip. In the calculation of mean levels of prostaglandin-like activity, all samples with subthreshold activity were assigned the threshold value of the assay, namely 3 p-mole/l. (1 ng/ml.). Because this assumption might have biased the statistics, all the calculations were repeated assigning subthreshold samples zero concentration. The only mean values that were altered significantly were those of the samples collected before time zero (see Fig. 1). None of the conclusions was affected. When c.s.f. from other rabbits receiving the same pyrogen was extracted and separated using thin-layer chromatography, the active ingredient moved with prostaglandins of the E series. This identification was confirmed by radio-immunoassay.
W. I. CRANSTON AND OTHERS DISCUSSION
Our experiments show: first, when a plateau fever is produced by the infusion of endogenous pyrogen, PGE levels in cisternal c.s.f. increase, also to a plateau; secondly, the additional infusion of an appropriate dose of salicylate prevents the release of PGE into c.s.f. but does not affect the development of the fever. It is now well established that the fever produced by endotoxin is normally associated with prostaglandin release into c.s.f. (see Dey et al. 1974), as is the fever caused by the virus of Newcastle Disease (PhilippDormston & Siegert, 1974b). Philipp-Dormston & Siegert (1974b) were the first to show that the fever produced by endogenous pyrogen is also associated with PGE release and our experiments confirm their findings quantitatively. We believe our experiments to be the first in which the time course of prostaglandin release has been followed during a steady-state fever. The level reached in cisternal c.s.f. 30 min after the administration of pyrogen was maintained over a further 3 hr of infusion. We do not attach great significance to the quantitative aspects of this observation. The level of prostaglandin in cisternal c.s.f. will depend not only on the rate of prostaglandin release into the c.s.f. but also on such factors as the rate of c.s.f. production, the rate of loss due to sampling, and the patterns of c.s.f. flow in and around the brain, all of which could change during the course of the experiment. We can conclude, however, that throughout the fever there was a release of prostaglandin into the c.s.f. The administration of salicylate in our experiments did not significantly change the latency, rate of rise, or level of the fever produced by pyrogen administration. The failure of the salicylate to produce antipyresis confirms earlier reports of its weak antipyretic properties in rabbits (Grundman, 1969; Cranston et al. 1970, 1971; van Miert et al. 1972). We have now shown that in spite of its failure to produce antipyresis, the salicylate prevented the appearance of prostaglandin in c.s.f. samples from the cisterna. The fact that it is possible to dissociate fever from prostaglandin release vitiates previous arguments in which the elevation of prostaglandin levels in cisternal c.s.f. has been used to implicate prostaglandin as a necessary causal link in fever. Similarly, the argument that aspirin-like drugs can prevent the appearance of prostaglandin in cisternal c.s.f. may no longer be used as evidence that the mechanism of antipyresis of the aspirin-like drugs is inhibition of prostaglandin synthesis. The prostaglandin in cisternal c.s.f. during fever is likely to originate from widespread areas of the brain (Feldberg et al. 1973). One possible explanation for our results would be that the salicylate, while preventing
591 FEVER AND PROSTAGLANDIN RELEASE the appearance of prostaglandin in c.s.f., did not reduce the hypothalamic concentrations sufficiently to affect the fever. Though possible, this explanation is unlikely. The dose of salicylate used was similar to a dose shown in previous experiments (Rawlins et al. 1973) to produce concentrations of salicylate in the hypothalamus sufficient to inhibit prostaglandin synthesis by at least 50 %. Since the dose of leucocyte pyrogen used in our experiments was submaximal (cf. Cranston et al. 1971) partial inhibition of prostaglandin synthesis might have been expected to cause partial abatement of the fever; none was observed. Veale & Cooper (1975) have recently reported that it is possible to create lesions in the brains of rabbits which render them insensitive to the hyperthermic effects of prostaglandins; they still develop fever in response to pyrogen. Also, the prostaglandin antagonist SC 19220 has been reported to block prostaglandin fevers, but not pyrogen fevers (Sanner, 1974). These observations, together with the results of our experiments, suggest that the idea that prostaglandin synthesis forms an essential link in the action of pyrogens on the brain must be called into question. We are grateful to W. Feldberg, F.R.S., for his encouragement and advice; to M. Dashwood for his painstaking assay of all the c.s.f. samples; to Susan Cammock and A. S. Milton for characterizing the prostaglandin and for commenting on the MS; to Margaret Tester for assistance with the experiments; to J. E. Pike and the Upjohn Company for supplying prostaglandin samples; and to the Pharmacy, St Thomas's Hospital, for supplying sterile salicylate solutions. REFERENCES
ATKINS, E. & BODEL, P. T. (1971). Role of leucocytes in fever. In Pyrogen8 and Fever, ed. WOLSTENHOLME, G. E. W. & BIRCH, J. pp. 81-98. Edinburgh: Churchill Livingstone. CRANSTON, W. I., HELLON, R. F. & MITCHELL, D. (1975). Fever and brain prostaglandin release. J. Phy8iol. 248, 27-29P. CRANSTON, W. I., LUFF, R. H., RAWLINS, M. D. & ROSENDORFF, C. (1970). The effects of salicylate on temperature regulation in the rabbit. J. Phy8iol. 208, 251-259. 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. & STITT, J. T. (1975). Behavioural and autonomic induction of prostaglandin E1 fever in squirrel monkeys. J. Physiol. 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. Physiol. 241, 629-646. DozoIs, R. R. & THOMPSON, C. J. (1974). Presence of prostaglandin E2 and A2 in canine gastric solution. Life Sci., Oxford 15, 975-986. FELDBERG, W. & GUPTA, 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. Physiol. 234, 279-303. 20
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W. I. CRANSTON AND OTHERS
FELDBERG, W. & SAxENA, P. N. (1971 a). Fever produced by prostaglandin E1. J. Phyeiol. 217, 547-556. FELDBERG, W. & SAXENA, P. N. (1971 b). Further studies on prostaglandin E1 fever in cats. J. Phyaiol. 219, 739-745. FLOWER, R. J. (1974). Drugs which inhibit prostaglandin biosynthesis. Pharmac. Rev. 46, 33-67. GRIuNDMAN, M. J. (1969). Studies on the action of antipyretic substances. D.Phil. Thesis, University of Oxford. HALES, J. R. S., BENNETT, J. W., BAIRD, J. A. & FAWCETT, A. A. (1973). Thermoregulatory effects of prostaglandins E1, E2, F,. and F2a in the sheep. Pflugerq Arch. ge8. Phyeiol. 339, 125-133. HELLON, R. F. (1974). Monoamines, pyrogens and cations: their actions on the central control of body temperature. Pharmac. Rev. 46, 289-321. 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. Phyeiol. 218, 325-336. PHLPP-DORMSTON, W. K. & SIEGERT, R. (1974a). Identification of prostaglandin E by radioimmunoassay in cerebrospinal fluid during endotoxin fever. Naturwmien8chaften 61, 134-135. 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, W. J. & EAST, P. F. (1972). Effects of prostaglandin E2 on the body temperature of conscious rat and cat. Arch8 int. Pharmacodyn. Thdr. 197, 31-36. RAWLiNS, M. D., LUFF, R. H. & CRANSTON, W. I. (1973). Regional brain salicylate concentrations in afebrile and febrile rabbits. Biochem. Pharmac. 22, 2639-2642. SANNER, J. H. (1974). Substances that inhibit the actions of prostaglandins. Archs intern. Med. 133, 133-146. STITT, J. T. (1973). Prostaglandin E1 fever induced in rabbits. J. Phyeiol. 232, 163-179. VAN MIERT, A. S. J. P. A. M., VAN ESSEN, J. A. & TRoMP, G. A. (1972). The antipyretic effect of pyrazolone derivatives and salicylates on fever induced with leukocytic or bacterial pyrogen. Arch8 int. Pharmacodyn. Th&r. 197, 388-391. VEALE, W. L. & COOPER, K. E. (1975). Comparison of the sites of action of prostaglandin E and leucocyte pyrogen in brain. In Temperature Regulation and Drug Action, ed. LoMAX, P., SCHONBAum, E. & JACOB, J., pp. 218-226. Basel: Karger.