(Received 31 January 1973)

Transcription

1 J. Phygiol. (1973), 234, pp With 6 text-figures Printed in Great Britain EFFECT OF PYROGEN AND ANTIPYRETICS ON PROSTAGLANDIN ACTIVITY IN CISTERNAL C.S.F. OF UNANAESTHETIZED CATS BY W. FELDBERG, K. P. GUPTA,* A. S. MILTONt AND SABINE WENDLANDTt From the National Institute for Medical Research, Mill Hill, London NW7 1AA (Received 31 January 1973) SuMMARY 1. Samples of cisternal cerebrospinal fluid (c.s.f.) were collected from unanaesthetized cats while rectal temperature was continuously recorded. From the same cat, samples were collected during normal body temperature, during pyrogen fever and when the fever was brought down by an i.p. injection of an antipyretic. Fever was produced by injection of the bacterial pyrogen of Shigella dysenteriae either into the third ventricle, cisterna magna or i.v. The samples of c.s.f. were assayed for PGE1-like activity on the rat stomach fundus strip preparation rendered insensitive to 5-HT. 2. In samples of c.s.f. collected during normal body temperature, usually either no PGE1-like activity was detected, or its activity was low. Higher values were obtained in only a few cats. 3. In each experiment the PGE1-like activity increased, often many-fold, in samples collected during the pyrogen fever, irrespective, of the route of administration of the pyrogen. However, on i.v. injection, about 1000 times larger doses of the pyrogen were required than on injection into the liquor space to produce fever and the increase in PGE1-like activity of cisternal c.s.f. 4. The antipyretic drugs indomethacin, paracetamol and aspirin, injected i.p. during the pyrogen fever, brought down temperature, and the PGE1-like activity of the cisternal c.s.f. again became low. 5. When samples of cisternal c.s.f. were subjected to thin layer chromatography the prostaglandin-like activity was solely or mainly found in the zone corresponding to the prostaglandins of the E series. * Commonwealth Medical Fellow and later Wellcome Research Fellow. t Permanent address: Department of Pharmacology, University of Aberdeen. 4 Supported by a Wellcome Research Grant.

2 280 W. FELDBERG AND OTHERS 6. These findings support the theory that pyrogens produce fever by increasing synthesis and release of prostaglandin in the preoptic anterior hypothalamic area, and that antipyretics of the aspirin type bring down this fever because they inhibit this synthesis. 7. It is concluded that pyrogen increases prostaglandin synthesis not only in the preoptic anterior hypothalamic area. When injected into the liquor space increased synthesis of prostaglandin probably occurs in many regions near the surface of the brain stem, and when injected i.v. may occur in other parts of the C.N.s. as well. But to produce fever the prostaglandin has to act on the preoptic anterior hypothalamic area. INTRODUCTION In recent experiments on unanaesthetized cats, evidence was obtained that fever produced by a bacterial pyrogen was associated with an increase in prostaglandin-like activity of cerebrospinal fluid (c.s.f.) (Feldberg & Gupta, 1972, 1973). In these experiments, a Collison cannula had been implanted into the third ventricle in such a way that its tip lay rostral and ventral to the massa intermedia. The injection of pyrogen was made through this cannula, and also the collection of the c.s.f. which had therefore been in close contact with that part of the ventricular walls which contains the nuclei of the anterior hypothalamus. The prostaglandin-like activity of the ventricular c.s.f. again became low after an i.p. injection of the antipyretic paracetamol. This antipyretic brings down the fever produced by an intraventricular injection of pyrogen (Milton & Wendlandt, 1968). In the present experiments it is shown that the increase in prostaglandinlike activity of c.s.f. during pyrogen fever occurs also in c.s.f. collected from the cisterna magna, and that it occurs whether the pyrogen is injected into the third ventricle, the cisterna magna or i.v. Further, not only paracetamol but also indomethacin and aspirin injected intraperitoneally brought down pyrogen fever, and the prostaglandin-like activity again became low. Finally, when samples of c.s.f. were subjected to thin layer chromatography the prostaglandin-like activity was found solely or mainly in the zone corresponding to the prostaglandins of the E series. Some of the results were communicated to the British Pharmacological Society (Feldberg, Gupta, Milton & Wendlandt, 1972). METHODS Cats weighing between 3 and 3-5 kg were used. The method for collecting c.f.s. was that described for collecting effluent from the cisterna magna on perfusion of the ventricular system in unanaesthetized cats (Feldberg, Myers & Veale, 1970). In an

3 FEVER AND CISTERNAL PROSTAGLANDIN 281 aseptic operation under pentobarbitone sodium anaesthesia, a Collison cannula, with a shaft 12 mm long, was fixed to the back of the skull with acrylic cement, at an angle of 48 from the vertical, so that the tip of the shaft rested just above the atlanto-occipital membrane. The method of fixing the cannula has been described in detail by Feldberg et al. (1970). In Fig. IA, the position of the cannula is shown in the diagram of a midsagittal section of the cat's brain. As cats have no foramen of Magendie, the ventricular c.s.f. does not pass into the cisterna magna from the caudal end of the fourth ventricle, but enters the subarachnoid space solely through the foramina of Luschka at the lateral recesses of the fourth ventricle. To indicate the absence of a direct pathway from the caudal end of the fourth ventricle to the cisterna magna, the ventricular space is shown in the diagram in black, and the subarachnoid space by dotted areas. A B Fig. 1. A: diagram of midsagittal section of cat's brain with Collison cannula fixed to the back of the skull. Black area: ventricular space. Dotted areas: subarachnoid space. Striped area surrounding the cannula: acrylic cement. M.I.: massa intermedia. B: diagram of Collison cannula with hollow needle inserted. The screw at the side of the cap holds the needle in place after it has entered the cisterna magna. After the cat had recovered from the operation, samples of c.s.f. were collected without anaesthesia. For this purpose, the cap of the cannula was unscrewed and replaced by another cap modified by A. R. J. Collins. It had a screw inserted into its side, as illustrated in diagram B of Fig. 1. Before screwing on this cap, its rubber diaphragm was pierced by a hollow stainless-steel needle (gauge 28). Attached to the protruding outer end of the needle was a length of fine polyethylene tubing connected to a 1 ml. syringe filled with 0 9 % NaCl solution. After filling tubing and needle with the solution, the cap was screwed on and the needle lowered into the shaft of the cannula until it met with some resistance, which meant it had touched the atlanto-occipital membrane. To pierce the membrane the needle was sharply lowered mm. On disconnecting the syringe and keeping the free end of the tubing a few cm below the level of the head of the cat, drops of clear c.s.f. flowed out at a rate of ml./min. Once the flow was established the screw at the side of the cap was tightened. This prevented shifting of the needle tip inside the cisterna magna during collecting and in the intervals between collections. Samples of ml. c.s.f. were collected at intervals of 1-4 hr. Between collections, loss of c.s.f.

4 282 W. FELDBERG AND OTHERS from the tubing was prevented by plugging its free end with a pin, and attaching the tubing with adhesive tape to the head of the cat. The samples of c.s.f. were assayed either immediately after the collection, or after having been stored at - 20 C for hr. Their activity was assayed in terms of PGEL on the rat's stomach fundus strip preparation rendered insensitive to 5- hydroxytryptamine by adding 1 mg methysergide, dissolved in ml. methanol, to the 5 1. Krebs solution in the reservoir. Further, 5 mg indomethacin, dissolved in ml. ethanol, was added to the reservoir to ensure relaxation of the muscle and to prevent spontaneous increases in tone. Indomethacin inhibits synthesis of prostaglandins (Vane, 1971). It has been shown that synthesis and release of prostaglandins occur in the rat's stomach wall (Coceani & Wolfe, 1966; Bennett, Friedmann & Vane, 1967; Pace-Asciak, Morawska, Coceani & Wolfe, 1967) and that the tone of some isolated smooth muscle preparations, including that of the rat stomach fundus strip, is maintained by intramural generation of prostaglandins (Bennett & Posner, 1971; Ferreira, Herman & Vane, 1972). The sensitivity of the preparation to prostaglandins either remains unaffected or is increased by the addition of indomethacin to the bath fluid (Eckenfels & Vane, 1972). In the present experiments the sensitivity varied greatly. With some preparations as little as 0 3 ng PGE1 could be detected in 1 ml. c.s.f., in others only 3 ng. Rectal temperature was recorded at room temperature (21-23 C) with a thermistor probe (Yellow Spring Instrument Co.) inserted about 10 cm deep into the rectum and held in position by adhesive tape attached to the protruding end of the probe and gently wrapped around the base of the tail. Temperature was monitored continuously by a Kent recorder, and the Figures reproduced are plotted directly from the tracings obtained in this way. The pyrogen used was Shigella dysenteriae somatic type 0 antigen from the World Health Organisation Pyrogen Standard (Humphrey & Bangham, 1959). For the injections into the third ventricle and into the cisterna magna it was dissolved in pyrogen-free artificial c.s.f., and for the i.v. injections in pyrogen-free 0-9% NaCl solution. The injections into the third ventricle (75 ng in 0O15 ml.) were made through a Collison cannula implanted into the ventricle rostral to the massa intermedia as described previously (Feldberg & Gupta, 1973). The cannula was implanted at the same time as the cisternal cannula was fixed to the back of the skull. The injections into the cisterna magna (75 ng in 0-15 ml. or 150 ng in 0 3 ml.) were made through the hollow needle inserted into the cisternal cannula for collection of c.s.f. Intravenous injections of the pyrogen were made into the brachial vein of a foreleg. Thin layer chromatography. Samples of cisternal c.s.f. collected before, during and after fever were submitted to thin layer chromatography according to the method described by Horton & Main (1967). Commercially prepared silica coated sheets ('Chromagram' Eastman) of 20 cm x 20 cm were used. On each sheet 2 mm wide bands of the silica were scraped off so as to obtain four to six strips of equal width. The sheet was then activated in an oven at C for min. Samples of ml. c.s.f. obtained from one to three experiments were each made up to 5 ml. with artificial c.s.f. brought to ph 3-5 with 0-1 N-HCl and partitioned twice with 10 ml. ethyl acetate. The aqueous phase was discarded and the ethyl acetate phase was evaporated to dryness under reduced pressure on a rotary evaporator in a water-bath at 400 C. The residue of each sample was dissolved in 50,u. methanol, drawn into a capillary tube and applied in a series of spots, about 2 cm from the bottom edge, to a strip covering its entire width. PGE, dissolved in artificial c.s.f. (20-50 ng in 5 ml.) was similarly extracted and applied in the same way to the bottom of another strip. Sometimes one strip was used for PGF2<, treated in the same way as PGE,, and one strip was run as a solvent blank. One strip always

5 FEVER AND CISTERNAL PROSTAGLANDIN 283 served as a marker strip by applying about 2 cm from its bottom edge approximately 20 du. methanol solutions of PGE1 (1 mg/ml.) and of PGF2a (0.5 mg/ml.), each as a separate spot. The sheet was run in a 'Chromagram' chamber plate set (Eastman). To separate the prostaglandins of the E series from those of the F series, the Al solvent system of Gr6en & Samuelsson (1964) was used. It contains benzene, dioxane and acetic acid in a proportion of 20:20: 1. When the solvent had reached cm above the origin, the sheet was removed from the chamber, the position of the solvent was marked and the sheet was dried in a stream of air at room temperature from a hair dryer. The marker strip was cut off, sprayed with 10 % phosphomolybdic acid in ethanol. On heating the strip in an oven at ' C for a few minutes, the prostaglandins appeared as grey-blue spots on a greenish-yellow background. The other strips were divided into zones corresponding to the B. values on the marker strip for prostaglandin E1 (0.66 to 0*68) and F2. (0.52). From each strip the E and F zones were scraped off. The scrapings from each zone of each strip were extracted separately by shaking them with 5 ml. methanol in a centrifuge tube; they were then centrifuged, re-extracted with a further 5 ml. methanol and again centrifuged. The combined methanol extracts were evaporated to dryness under reduced pressure on a rotary evaporator in a water-bath at 400 C. The residue was dissolved in 0-9 % NaCl solution and assayed on the rat stomach fundus strip preparation as PGE1, except when recovery of PGF2, was measured, then the assay was as PGF2,. Drugs. Paracetamol (4-acetamidophenol) (Koch, Light) was dissolved in a hot 0 9 % NaCl solution containing 20% propylene glycol as described by Milton & Wendlandt (1971); 50 mg/kg were injected i.r. in a volume of 4 ml. Indomethacin was kindly supplied by Merck, Sharp & Dohme, Hoddesdon, Herts. A 2 mg/ml. solution in 0-9 % NaCl was made by the addition of a small amount of sodium bicarbonate. Of this solution, 1 ml./kg was injected i.r. Aspirin (acetylsalicylic acid) (BDH) was dissolved by adding 0 4 g to 10 ml. 8 % potassium citrate. The solution was diluted with 0-9 % NaCl, so that 4 ml. contained the amount of aspirin to be injected i.p., i.e. 25 mg/kg. PGE1 and PGF21 were kindly supplied by Dr John Pike (Upjohn Co., Kalamazoo, Michigan, U.S.A.) and stock solutions containing 100,ug/ml. were prepared as described elsewhere (Milton & Wendlandt, 1971). Methysergide bimaleate (kindly supplied by Sandoz Products Ltd). Values refer to the salt. RESULTS In sixty-five experiments on thirty-nine cats which had no fever (temperature between 38-3 and C), cisternal c.s.f. was collected before an injection of pyrogen was made. In the samples from forty experiments the PGEl-like activity was either less than 1 ng/ml. c.s.f., or below the threshold of sensitivity of the rat fundus strip preparation. In twenty-one of the other twenty-five experiments, the activity of the c.s.f corresponded to between 1 and 3 ng/ml., and in the remaining four to 5, 6, 12 and 17 ng/ml., although these cats had no fever. When c.s.f. was collected from the same cat on different days, the PGE1-like activity was not always the same. For instance, in the one cat with a PGEl-like activity of 6 ng/ml., none was detected in its c.s.f. collected a few days later. Cisternal c.s.f. was collected from two cats which had fever, 40-2 and

6 284 W. FELDBERG AND OTHERS C, of unknown aetiology; the PGE1-like activity of the c.s.f. corresponded to 5 ng/ml. in the one, and to 30 ng/ml. in the other. Pyrogen injected into the third ventricle An injection of 75 ng of the bacterial pyrogen of Shigella dysenteriae produced fever which lasted for several hours. During the fever, the cat sat in its cage with its head lowered, eyes closed, and its forepaws tucked under its body. The cat often appeared stuporous. The fever was associated with an increase in PGE1-like activity of the cisternal c.s.f. Following an intraperitoneal injection of an antipyretic, fever was brought down, the PGE1-like activity again became low, and the cat recovered from its stupor. The upper record of Fig. 2 illustrates an experiment in which the PGE1- like activity of the c.s.f. increased from < 1 ng/ml. to 10 ng/ml. during the fever, and was again < 1 ng/ml. when the fever was brought down by paracetamol. Table 1 summarizes the results from six experiments. Before the pyrogen injection, the PGE1-like activity of the c.s.f. was between 0 and 2-5 ng/ml.; hr after the injection, when temperature had risen to between 40-2 and 420 C it was between 5 and 20 ng/ml., and when temperature had fallen to between 38'7 and C, 1P5-2 hr after the injection of either indomethacin or paracetamol, it was again low, 1 ng/ml. or less. In Expt. 6 of Table 1, in which the pyrogen had increased the PGE1-like activity from 2-5 to 20 ng/ml., it decreased to 2 ng/ml. in a third sample collected on the next day when fever had subsided. Pyrogen injected into the cisterns magna An injection of the pyrogen into the cisterna magna produced fever, an increase in the PGE1-like activity of cisternal c.s.f. and behavioural effects similar to those that occurred after injection of pyrogen into the third ventricle. The lower record of Fig. 2 shows an experiment in which 75 ng were injected into the cisterna magna. The fever and the increase in PGE1-like activity were similar to those of an injection of 75 ng into the third ventricle. In thirty-five other experiments in which the dose of pyrogen injected was 150 ng, temperature rose to between 39-9 and C. The fever lasted for several hours, and the PGE1-like activity in the c.s.f. which before the injection was between 0 and 12 ng/ml. (mean 1-3 ng/ml.), had increased hr after the injection, to between 4 and 100 ng/ml. (mean 22*8 ng/ml.). The increase in PGE1-like activity occurred already during the steep rise in temperature which began min after the injection. In one experiment in which temperature had risen to over 410 C within 50 min and was still rising, the activity of c.s.f. collected at this time was as high as

7 FEVER AND CISTERNAL PROSTAGLANDIN ng/ml., compared with < 2 ng/ml. in the control sample collected before the injection. In five experiments, 150 ng of the pyrogen was injected twice at an interval of 1*5 hr without collecting c.s.f. in between, because the first injection had not produced a great rise in temperature. The PGE1-like activity increased from between 0 and 0-25 ng/ml. to between 6 and oc 4 Hours. Fig. 2. Records of rectal temperature from two unanaesthetized cats. The height of the columns and the values above refer to PGE1-like activity in ng/ml. of cisternal c.s.f., the position of the columns refers to the time but not the duration of the c.s.f. collection. In the top record the first arrow indicates injection of 75 ng pyrogen into the third ventricle and the second arrow i.p. injection of paracetamol 50 mg/kg (Para). In the bottom record the first arrow indicates injection of 75 ng pyrogen into the cisterna magna, and the second arrow i.p. injection of indomethacin 2 mg/kg (Indom'n).

8 286 W. FELDBERG AND OTHERS 75 ng/ml. (mean 31 ng/ml.) in the samples collected 15-3 hr after the second injection. There was no relation between the initial PGE1-like activity in the control samples and the activity in the fever samples. In two experiments in which no PGEl-like activity was detected in the control samples the activity of the fever samples corresponded to 4 ng/ml. in the one, and to 100 ng/ml. in the other; and in a third experiment with a control value of 6 ng/ml., it increased to 16 ng/ml. There was also no relation between the degree of fever and PGE1-like activity. For instance, in one experiment, temperature rose to C and PGE1-like activity increased to 100 ng/ml.; in another, temperature rose to C and PGE1-like activity increased to 18 ng/ml.; and in yet another, temperature rose to C only, but PGEl-like activity increased to 83 ng/ml. It was stated (page 284) that in one cat which had fever (40.70 C) of unknown aetiology the PGE1-like activity of its c.s.f. was 30 ng/ml. Two injections of 150 ng pyrogen raised temperature to C and the PGE1- like activity of the c.s.f. increased to 120 ng/ml. When two samples of c.s.f. were collected after an intracisternal injection of 150 ng of the pyrogen, the PGE1 equivalent in the second sample collected hr after the first, while temperature was still high, was usually greatly reduced. This is illustrated in the Expt. Fig. 3 (middle record) and the results of eight experiments are summarized in Table 2. In these, the mean reduction in PGE1-like activity was from 17*8 to 8-4 ng/ml. On the other hand, when another 150 ng of the pyrogen was injected between the collection of the two fever samples, there was either only a slight reduction, as in the experiment of the upper record of Fig. 3, or a further increase in PGEl-like activity. Table 3 summarizes the results of eight experiments, including five in which such an increase occurred. Taking the mean values of all eight experiments, there was an increase from 12-1 to 20*1 ng/ml. as compared to the decrease from 17-8 to 8-4 ng/ml. in the experiments of Table 2. However, when a third fever sample was collected 1-2 hr after the second one, the PGE1-like activity decreased to the same extent as in the experiments of Table 2 between the first and second fever samples. This is illustrated in Fig. 3 (upper record) and is shown for five of the experiments of Table 3 (last column). In c.s.f. collected 24 hr after a pyrogen injection, when fever had subsided, the PGEl-like activity was again low, 2 ng/ml. or less. Antipyretics Their i.p. injection during pyrogen fever brought down the fever, abolished the stuporous state, and reduced the high PGE1-like activity of the cisternal c.s.f. The effect of indomethacin (2 mg/kg) is illustrated in

9 FEVER AND CISTERNAL PROSTAGLANDIN ng shig. cist c la I I u I I v-- I I Hours I 8 9 Fig. 3. Records of rectal temperature from three unanaesthetized cats. The height of the columns and the values above or beside them refer to PGEIL-like activity in ng/ml. of cisternal c.s.f., the position of the columns refers to the time but not the duration of the c.s.f. collection. All arrows, except the second one in the bottom record, indicate injections of 150 ng pyrogen into the cisterna magna. The second arrow in the bottom record indicates an i.r. injection of paracetamol 50 mg/kg (Para).

10 288 W. FELDBERG AND OTHERS Fig. 2 (bottom record), that of paracetamol (50 mg/kg) in Fig. 3 (bottom record), and that of aspirin (25 mg/kg) in Fig. 4 (top record). The results of seven experiments are summarized in Table 4. From the mean values it is evident that the decrease is from 25'3 to 3-1 ng/ml. Without the intervening injection of an antipyretic, the decrease was much less. For TABLE 1. Rectal temperature and PGE.l-ike activity of cisternal c.s.f. (A) before, (B) 2-3*5 hr after an injection of 75 ng pyrogen into the third ventricle, and (C) hr after an i.p. injection of either indomethacin 2 mg/kg (Indom'n) or of paracetamol 50 mg/kg (Para) PGE1-like activity in c.s.f. Temperature (0C) (PGE1 ng/ml.) AKr A I Expt. A B C A B C (< 0.3) 5 1 Indom'n (< 0.7) Indom'n 4* (< 1) 10 0(< 1) Para *6 0(<2) * Mean * Same experiment as top record of Fig. 2. TABLE 2. PGE-like activity in cisternal c.s.f. collected before (A) and during (B and C) fever produced by an injection of 150 ng pyrogen into the cisterna magna. Samples B collected hr after the pyrogen injection; samples C collected hr after B PGE1-like activity in c.s.f. (PGE1 ng/ml.) Expt. A B C ' * 0(< 1) Mean * Same experiment as middle record of Fig. 3. instance, the mean values fell from 17-8 to 8.4 ng/ml. between the first and second fever samples in the experiments of Table 2, and from 20 1 to 9 ng/ml. between the second and third fever samples in the experiments of Table 3. Two of the antipyretics, indomethacin and paracetamol, were injected I.P., min before the pyrogen; they prevented or delayed the rise in

11 FEVER AND CISTERNAL PROSTAGLANDIN 289 temperature and the increase in PGE1-like activity of c.s.f. This is illustrated by the two experiments of Fig. 5. In the one (top record) with indomethacin, there was only a small delayed rise in temperature without any detectable increase in PGE1-like activity. In the other, with paracetamol, the pyrogen produced a delayed rise in temperature which began about 5 hr after its injection; the PGE1-like activity increased to 3 and 2 ng/ml. in the first two samples; the further increase to 5 ng/ml. in the third sample collected about 8.5 hr after the pyrogen injection when temperature had risen to C was probably a sign that the main effect TABLE 3. PGE1-like activity in cisternal c.s.f. collected before (A) and during (B to D) fever produced by pyrogen injected intracisternally. B collected hr after first injection of 150 ng of the pyrogen, C collected 2-3 hr after second injection of 150 ng of the pyrogen which was made Inin after collection of B. D collected 1-2 hr after C (PGE1 ng/ml.) PGE1-like activity in c.s.f. Expt. A B C D 1 0(< 1) (< 2) (< 2) (< 1.5) (< 2) (< 2) * Mean * Same experiment as top record of Fig. 3. of the paracetamol was over. Table 5 summarizes the results of eight experiments. From the mean values it is seen that the PGE1-like activity increased from 0-8 to only 2-6 ng/ml. in the c.s.f. collected hr after the pyrogen injection. Pyrogen injected intravenously An i.v. injection of the pyrogen produced fever and an increase in the PGE1-like activity of the cisternal c.s.f., but to produce these effects, about 1000 times larger doses had to be injected than on injection into the liquor space. The behavioural effects were similar to those following the injection into the liquor space, but in addition, vomiting and defaecation occurred. With successive samples of c.s.f. collected during fever, the PGEl-like activity often increased, instead of decreasing, as it usually did in the experiments when the pyrogen was injected intracisternally. In two experiments, 40,ug pyrogen were injected i.v. Temperature rose to just over 400 C, and the PGE1-like activity increased from 0 to between 1-2 and 1.5 ng/ml. PGE1 in the c.s.f. collected 2-4 hr after the injection.

12 290 W. FELDBERG AND OTHERS Following an i.v. injection of 250,ug pyrogen, the increase in PGE1-like activity varied from experiment to experiment. In some, the activity increased little more than after an i.v. injection of 40,ug. The results of eight such experiments are summarized in Table 6. In none of them did the PGE1-like activity exceed 3 ng/ml. On the other hand, in eleven experiments, summarized in Table 7, values from 7-5 to 195 ng/ml. were reached. These differences could not be related to the degree of fever. True, in three 150 ng Aspirin 41 shig. cist. 25 mg/kg shig.cis.v.,~ 2 g ll l l l l ugs 38 1 lla 4.5 ~ ~ ~ Hours Fig. 4. Records of rectal temperature from two unanesthetized cats. The height of the columns and the values above refer to PGFE1-like activity in ngfml. of cisternal c.s.f., the position of the columns refers to the time but not the duration of the c.s.f. collection. In the top record the first and second arrows indicate injections of 150 ng pyrogen into the cisterna magna, and the third arrow indicates an I.iP. injection of aspirin 25 mg/kg. In the bottom record the first arrow indicates an I.v. injection of 250,ug pyrogen. and the second arrow an x.i. injection of aspirin 25 mg/kg.

13 FEVER AND CISTERNAL PROSTAGLANDIN 291 experiments of Table 6 (nos. 3, 4 and 8), fever did not rise above 400 C, but in the others, fever was of the same magnitude as in most experiments of Table 7. Further, the highest values, 195 and 60 ng/ml., were obtained in samples collected when temperature was 40 4 and C (Expts. 5 and 8 of Table 7), whereas in five samples collected at a time when temperature was above 410 C the PGE1-like activity corresponded to 2*2, 7-7, 3-6, 13 and 17 ng/ml. (Expt. 6 of Table 6, Expts. 1, 3 and 4 of Table 7). 41 r- L 150 ng shig. cist C r ng shig. cist Hours Fig. 5. Records of rectal temperature from two unanaesthetized cats. The height of the columns and the values above refer to PGE1-like activity in ng/ml. of cisternal c.s.f., the position of the columns refers to the time but not to the duration of the c.s.f. collection. The first arrow indicates an i.p. injection of indomethacin 2 mg/kg (Indom'n) in the top, and of paracetamol 50 mg/kg (Para) in the bottom record. The second arrow indicates in each record an injection of 150 ng pyrogen into the cisterna magna.

14 292 W. FELDBERG AND OTHERS The results of three experiments of Table 7 are given in detail in Figs. 4 and 6. They illustrate that the progressive increase in PGE1-like activity, in successive samples collected during fever, occurs whether temperature rises simultaneously or not. In the experiment of the bottom record of Fig. 4, the activity had increased from < 1 to 11 ng/ml. about 3 hr after the pyrogen injection when temperature was C, and about 2-5 hr later, it had further increased to 13 ng/ml. although temperature was 0.10 C lower. In the experiment of the top record of Fig. 6, three samples collected TABLE 4. PGE1.-ike activity in cisternal c.s.f. collected before (A), hr after (B), an intracisternal injection of 150 ng pyrogen and hr after (C), an i.p. injection of either indomethacin 2 mg/kg (Indom'n), paracetamol 50 mg/kg (Para), or aspirin 25 mg/kg. The antipyretics were injected min after collection of B PGEI-like activity in c.s.f. (PGE1 ng/ml.) C Expt. A B 1 0(< 3) 13 6 Indom'n Indom'n 3 1P Indom'n 4* 0 (< 0.3) Para Para 6 0(< 1) 8 3 Aspirin 7 0(< 1) 15*5t 1 Aspirin Mean * Same experiment as bottom record of Fig. 3. t Collected 1'5 hr after a second pyrogen injection. Same experiment as top record, of Fig. 4. TABLE 5. PGE1-like activity in cisternal c.s.f. collected before (A), and hr after (B), an intracisternal injection of 150 ng pyrogen which was preceded min earlier by an i.p. injection of either indomethacin 2 mg/kg (Indom'n) or paracetamol 50 mg/kg (Para) PGE1-like activity in c.s.f. (PGE1 ng/ml.) Expt. A B 1* 0 (< 1) 0 (< 1) Indom'n 2 0(< 0.7) 0-7 Indom'n 3 0 (< 1) 2-5 Indom'n 4 1P7 3.5 Indom'n 5 0 (< 1) 4-8 Indom'n Para 7* 0(< 1) 3 Para Para Mean * Same experiments as those of Fig. 5

15 FEVER AND CISTERNAL PROSTAGLANDIN 293 H rs ce r- * * *, ' Cot a) Eo 0 bdo 1- D) - - v v -- --a bo.5 O 4> * -t a).... d a 4a C) v - a) O - o-..i l v v v v v tr I _ V aw p4 CH e lo V-4 a: C) 0 O 0 0 a) *,.4 a) cia ce0 a1) 00 osh fh z P-1 CO 00 w 4 t-. pq 6(: C~( 6( 10 O4 m di 'a a)u.5 o 00 * 10 CO CO to IN 00 Co Co CO CO co CO CO fri -" N CO 10 CO

16 294 W. FELDBERG AND OTHERS C4-4,4 0 Q 00 I '-z r rt 04-1-: -5- I I I I - - * 0 *S tjbc 1- C4). C3.5 C) *5 C) 1j41 v~ D ft 00~~~ 0 _ d O *H o eq C3 04m H -- - e c o ~ o c) -r^ 0f 0 v I I X A CO CO -tco eq 1o 4 ~C) H 114 wi q I I I q; -O C) bc C).00 C) C t._,. C ḅ 0 fw (d Ek 140 o 00 c o-~o~oo,.d* *4 '" 00 aq C.) : " 1.01 " -Oq _ ;A 1-: 6 t eqs - C)0 *:'.I:. CO < O t O ~OCO.1+t co o oco t 1 W 2 *) CO es eq o o CO co csx cocs CO cco ct CO co Co CO o cococo E-Iq O cf0 o - c 1- eq * X

17 FEVER AND CISTERNAL PROSTAGLANDIN 295 about 2-5, 4 and 5 hr after the pyrogen injection, had a PGE1-like activity of 8, 17 and 20 ng/ml. but temperature was highest (41.20 C) when the second, and lowest (40 7 C) when the third fever sample was collected. On the other hand, in the experiment of the bottom record of Fig. 6, the increase in PGE1-like activity occurred together with a rise in temperature; it was 2x5 ng/ml. 2 hr after the pyrogen injection when temperature was C, and 12 ng/ml. 2 hr later when temperature was C. Whereas with intracisternal injections of pyrogen the increase in PGE1- like activity was obtained in samples of c.s.f. collected during the first 0c Hours. Fig. 6. Records of rectal temperature from two unanaesthetized cats. The height of the columns and the values above refer to PGEL-like activity in ng/ml. of cisternal c.s.f., the position of the columns refers to the time but not to the duration of the c.s.f. collection. The first arrow in both records indicates an i.v. injection of 250 #tg pyrogen, the second arrow in the bottom record, an i.r. injection of indomethacin 2 mg/kg (Indom'n).

18 ~W. FELDBERG AND OTHERS hour of the injection during the steep rising phase of temperature, this was not so with the ixv. injections. The increase occurred later and was not seen in samples of c.s.f. collected during the first hour of the pyrogen injection although temperature was already high. AntipyretiC8 Fever and the high PGE1-like activity of cisternal c.s.f. produced by an i.v. injection of the pyrogen, were abolished by antipyretics injected iap., and the cats recovered from their stuporous state. The effects of aspirin and indomethacin on temperature and PGE1-like activity are illustrated in Figs. 4 and 6. In the experiment of the bottom record of Fig. 4, aspirin lowered temperature from 40-2 to C, and the PGE,-like activity in c.s.f. decreased from 13 to 2-5 ngfml., and in the experiment of the bottom record Fig. 6, indomethacin lowered temperature from 40*7 to C and the PGE1-like activity decreased from 12 to < 1-5 ng/ml. Table 7 gives six experiments (nos. 6-11) in which indomethacin, paracetamol or aspirin, was injected after the second fever sample. The antipyretics brought down fever from a mean of 40-6 to C and the PGE1-like activity decreased from a mean of 15-6 to 1-9 ngfml., whereas in the five control experiments (nos. 1-5) the mean fall in temperature was from 40-6 to 40-3' C and the mean decrease in PGE1-like activity from 15-6 to 13-9 ng/ml. Thin layer chromatography of cisternal C.8.f. Fever samples of c.s.f. containing PGE1-like activity of between 12 and 41*5 ng were subjected to thin layer chromatography. The activity was found in the zone corresponding to the RF values of the prostaglandin of the E series. Table 8 shows the results for eight experiments. From a comparison of the values shown under B, at II, it is seen that in six experiments no activity was found in the F zone. In the other two, some activity was found in this zone as well, but this happened also for the standard solutions of PGE1. In fact, after chromatography of such solutions, some activity was found in the F zone in two further experiments (Expts. 4 and 7), suggesting that some of the PGE, had stayed in the F zone. In two experiments, nos. 5 and 7, one strip was run as a solvent blank. No activity was detected in either the E or F zones. In Expt. 7, one further strip was used for PGF2. (50 ng); in the F zone 20 ng were recovered but some activity was also detected in the E zone corresponding to 2 ng PGF2~. The recovery in the E zone of the PGE1-like activity of the fever samples varied between 40 and 77 % (mean 60-4 %), whereas the recovery of the

19 FEVER AND CISTERNAL PROSTAGLANDIN 297 b t 1 1 o 0 Z z " 1 ZZZZZ t~zch 0 0 ' bo bo o N~ 0 101X Ca [ q-4 m *00-4a 1-I '-" II T- cm la C~ t- w

20 298 W. FELDBERG AND OTHERS samples of the standard solutions of PGE1 in this zone varied between 35 and 100 % (mean 69-4 %). In the first four experiments of the Table, samples of c.s.f. collected before the pyrogen injection and containing between 0 and 4-5 ng PGE1-like activity were also subjected to thin layer chromatography. No activity was detected in either zone except in Expt. 2, in which for some unknown reason, 5 ng were found in the E zone. In Expt. 4, a third sample of c.s.f. collected after the fever had been brought down by aspirin was subjected to thin layer chromatography. Before chromatography, its PGE1-like activity corresponded to 1*5ng; after chromatography, 1 ng was recovered in the E zone and none in the F zone. DISCUSSION In experiments on unanaesthetized cats, it was recently found that the PGE1-like activity of c.s.f. collected from the third ventricle, i.e. c.s.f. which had probably been in close contact with the anterior hypothalamus, greatly increased during fever produced by injection of the bacterial pyrogen of Shigella dysenteriae into the third ventricle, and became low again when the fever was brought down by an intraperitoneal injection of paracetamol (Feldberg & Gupta, 1972, 1973). There was no need to have been so cautious about the origin of the c.s.f. because the same result has now been obtained with c.s.f. collected from the cisterna magna, not only when the pyrogen was injected into the third ventricle, but also when it was injected into the cisterna magna, or i.v. Further, the effect of bringing down the pyrogen fever and reducing the high PGE1-like activity of the c.s.f. was shown not only for paracetamol, but also for two other antipyretics, indomethacin and aspirin. All these results support the theory that pyrogens produce fever by increased synthesis and release of prostaglandins and that antipyretics bring down the fever because they inhibit prostaglandin synthesis as was shown by Vane (1971). As the rat stomach fundus strip preparation rendered insensitive to 5-HT contracts not only to PGE1, but also to PGE2, PGF1. and PGF2a (Coceani & Wolfe, 1966; Vane, 1969) the activity of the c.s.f. which was expressed in terms of PGE1 could have been due to any of the four prostaglandins, or to a mixture of them. However, only PGE1 and PGE2 produce fever when acting from the third ventricle on the anterior hypothalamus (Milton & Wendlandt, 1971), and the fact that the activity was found mainly, if not solely, in the E zone when samples of c.s.f. were subjected to thin layer chromatography, excludes a prostaglandin of the F series as the active substance in c.s.f. On the basis of mobility in silver impregnation thin layer chromatography, only PGE1 has been identified in cat's brain (Horton & Main, 1967). The activity found in cat's c.s.f. may there-

21 FEVER AND CISTERNAL PROSTAGLANDIN 299 fore have been due to this prostaglandin alone.* Combined gas chromatography-mass spectrometry on the t.l.c. eluate could be used to solve this problem. In dogs, all four prostaglandins have been detected in brain extracts (Holmes & Horton, 1968a) and in perfusate from the cerebral ventricles (Holmes, 1970), whereas in rabbit's brain, PGE2 has been detected, but not PGE1 (Ambache, Brummer, Rose & Whiting, 1966; Hopkin, Horton & Whittaker, 1968). It is therefore possible that in dogs both PGE1 and PGE2, and in rabbits only PGE2, may fulfil the role of mediator in the fever response to pyrogen. In a few of the control samples of c.s.f. collected from cats that had no fever, the activity was as high as, or even higher than, that found in fever samples. It is unlikely that it represents prostaglandin which has been stagnating in the subarachnoid space after having been released into this space for some time because Holmes & Horton (1968b) found that labelled PGE1 injected into the cerebral ventricles is rapidly removed from the c.s.f. However, as pointed out by Holmes (1970), there is a danger of obtaining wrong results when estimating prostaglandins in relatively impure extracts. He found that perfusate obtained from the perfused cerebral ventricles of dogs sometimes had a high PGE-like activity before, but none after silicic acid column chromatography. As the control samples of cat's c.s.f. with the high values were not subjected to thin layer chromatography (except one, and in this no activity was recovered) their activity might not have been due to prostaglandin. The possibility cannot be excluded that the very high prostaglandin-like activity of some of the fever samples may also not have been due entirely to prostaglandin, since these samples, too, were not subjected to thin layer chromatography. Whatever the explanation, there was regularly an increase of PGE-like activity during pyrogen fever and a decrease after the injection of an antipyretic, and these changes were still evident after thin layer chromatography. The origin of the PGE-like activity in the cisternal c.s.f. has so far not been definitely established. The high PGE-like activity found in fever samples is unlikely to represent prostaglandin that has been released into the third ventricle from the anterior hypothalamus and passed through the foramina of Luschka into the subarachnoid space because, due to dilution, the PGE-like activity would have been expected to be much lower in the cisternal than in the ventricular c.s.f. However, it was of the * From results recently obtained by Dr Heather Davis, Department of Pharmacology, University of Edinburgh, when she applied the method of radio-immunoassay to a pooled non-fever, and a pooled fever sample of cat's cisternal c.s.f., which we sent to her, it is clear that both the low activity of the non-fever sample and the high activity of the fever sample, is due mainly or wholly to PGE2 and not to PGE1.

22 300 W. FELDBERG AND OTHERS same order as that previously found in ventricular c.s.f. collected during pyrogen fever (Feldberg & Gupta, 1973). Probably the cisternal PGE-like activity represents prostaglandin that has been released into the subarachnoid space from the surface of the brain stem and of other parts of the brain. This implies that the action of pyrogen to increase synthesis and subsequent release of prostaglandin is not confined to the anterior hypothalamus and to the pre-optic region. Yet to produce fever, it has to act on these parts of the brain. Even when the pyrogen is injected into the third ventricle the release of prostaglandin may not be confined to the anterior hypothalamus because the pyrogen passes rapidly into the subarachnoid space through the foramina of Luschka. As cats have no foramen of Magendie, the fever produced by pyrogen injected into the cisterna magna is probably mainly due to its action on the preoptic region which, unlike the anterior hypothalamus, would be readily reached by pyrogen from the subarachnoid space, since it lies at the ventral surface ofthe diencephalon in close proximity to it. In this connexion it is interesting to note that PGE1 produces fever on intracisternal injection in doses which are only little larger than those effective on injection into the third ventricle (unpublished experiments). The idea that pyrogens increase synthesis and release of prostaglandin not only in the preoptic anterior hypothalamic area, poses the intriguing question of whether some of the symptoms of high fever, like malaise, are the result of high temperature. Instead, they may be effects produced by prostaglandin acting on parts of the C.N.s. other than this area. The pyrogen injections produced a stuporous state during which the cat did not react to events in its environment. Such a condition is also produced with PGE1 injected intraventricularly (Milton & Wendlandt, 1971) and when injected in doses much larger than those required to produce fever, catatonic stupor sometimes developed (Horton, 1964). The mechanism whereby fever and the rise in cisternal PGE-like activity are produced is probably not exactly the same when a bacterial pyrogen is injected intravenously, or into the liquor space. When injected intravenously, the bacterial pyrogen itself is not thought to act on the preoptic anterior hypothalamic area, but it first acts on the white cells of the host animal and releases from them leucocytic pyrogen, the 'ultimate pyrogenic agent' which acts on this region to elicit fever. Its action, according to the present experiments, would then be to increase synthesis and release of prostaglandin, not only in this region, but also in other parts of the c.n.s. and perhaps in other organs as well. On the other hand, when injected into the liquor space, the bacterial pyrogen may itself be the 'ultimate pyrogenic agent' that initiates the increased synthesis and release of prostaglandin, but this time only in those parts of the brain which lie in close

23 FEVER AND CISTERNAL PROSTAGLANDIN 301 proximity to the liquor space and are reached by the bacterial pyrogen when introduced into this space either by intraventricular or intracisternal injection. It would therefore appear that with the injections into the liquor space we do not imitate exactly the fever of infectious diseases, as we probably do on intravenous injection. A number of differences would readily be explained on these lines. For instance, why the increase of cisternal PGE-like activity should occur earlier after an intracisternal than after an i.v. injection, or why on collection of more than one sample of C.S.F. the PGE1 value of the second sample should so often be much lower than that of the first when the injection was made intracisternally, whereas the value usually remained high, or even increased in successive samples when the injection was made i.v. The reason is that if pyrogen injected intracisternally were to act locally on structures of the brain stem near the injection site, its effect should be greatest shortly after the injection when its concentration in the cisterna is highest. As the injected pyrogen spreads within the subarachnoid space, it becomes more and more diluted so that its action, although involving more distant parts of the brain, will become progressively weaker, and the initial concentration in the subarachnoid space is not attained again. Further, on removing a sample of c.s.f. it will be replaced by c.s.f. from more distant parts of the subarachnoid space to which the pyrogen has not spread, or only in a greatly weakened concentration. The replaced c.s.f. will therefore have a much lower PGE-like activity. In addition, in the removed c.s.f. some of the pyrogen will be removed as well, further reducing its concentration in the subarachnoid space. On the other hand, on i.v. injection, the increase in the synthesis of prostaglandin will occur gradually and will be progressive as the leucocytic pyrogen has first to be released and its release will continue to increase for some time. The increase in the synthesis of prostaglandin and consequently its release into the c.s.f. will therefore also occur gradually and be cumulative. From the finding that the PGE1 values of successive samples of c.s.f. collected over a period of several hours often increased, it can be postulated that during this time the prostaglandin synthesis goes on at an increasing rate either because the release of leucocytic pyrogen continues to increase, or because its release exceeds its destruction or inactivation. Further, if increased release of prostaglandin occurs from the surface of the brain stem and perhaps of the cerebrum as well, the replacement or removal of cisternal c.s.f. is not necessarily by c.s.f. with lower PGE-like activity. Finally, c.s.f. removal does not involve removal of any of the I.v. injected pyrogen or of the leucocytic pyrogen, and therefore no attenuation of the pyrogen effect will ensue. The three antipyretics examined, paracetamol, indomethacin and aspirin,

24 302 W. FELDBERG AND OTHERS all brought down pyrogen fever and reduced the high PGE1 values of cisternal c.s.f. This suggests that all three acted by inhibition of prostaglandin synthesis. Originally, Vane (1971) had found that paracetamol had a very weak inhibitory effect on prostaglandin synthesis in comparison to indomethacin and aspirin, although, like indomethacin and aspirin, it exerts strong antipyretic and analgesic action, yet it lacks their antiinflammatory effect. Vane's results were obtained on prostaglandin synthetase of guinea-pig's lung. Recently, Flower & Vane (1972) have reexamined the inhibitory effect of the three substances on brain synthetase of rabbit and dog because the antipyretic action and probably part of the analgesic action are central effects, whereas the anti-inflammatory action is a peripheral one. They found that paracetamol was about as active in inhibiting this synthetase as sodium aspirin. It would thus seem that antipyretics which lack an anti-inflammatory action, inhibit prostaglandin synthetase only in the c.n.s., but not in peripheral tissues. No discrepancy therefore exists between the results obtained with the three antipyretics examined in inhibiting prostaglandin synthesis in vitro and in bringing down pyrogen fever and the associated high PGE-like activity of cisternal c.s.f. in vivo. REFERENCES AMBACHE, N., BRuJMMER, HmARY C., ROSE, J. C. & WRITING, JUDITH (1966). Thin-layer chromatographyof spasmogenic unsaturated hydroxyacids fromvarious tissues. J. Physiol. 185, 77-78P. BENNETT, A., FRIEDMANN, C. A. & VANE, J. R. (1967). Release of prostaglandin EL from the rat stomach. Nature, Lond. 216, BENNETT, A. & POSNER, J. (1971). Studies on prostaglandin antagonists. Br. J. Pharmacy. 42, COCEANI, F. & WOLFE, L. S. (1966). On the action of prostaglandin E1 and prostaglandins from brain on the isolated rat stomach. Can. J. Physiol. Pharmac. 44, ECKENFELS, A. & VANE, J. R. (1972). Prostaglandins, oxygen tension and smooth muscle. Br. J. Pharmac. 45, FELDBERG, W. & GUPTA, K. P. (1972). Sampling for biological assay of cerebrospinal fluid from the third ventricle in the unanaesthetized cat. J. Physiol. 222, P. FELDBERG, W. & GUPTA, K. P. (1973). Pyrogen fever and prostaglandin activity in cerebrospinal fluid. J. Physiot. 228, FELDBERG, W., GUPTA, K. P., MILTON, A. S. & WENDLANDT, SABINE (1972). Effect of bacterial pyrogen and antipyretics on prostaglandin activity in cerebrospinal fluid of unanaesthetized cats. Br. J. Pharmac. 46, P. FELDBERG, W., MYERS, R. D. & VEALE, W. L. (1970). Perfusions from cerebral ventricle to cisterna magna in the unanaesthetized cat. Effect of calcium on body temperature. J. Physiol. 207, FERREIRA, S. H., HERMAN, A. G. & VANE, J. R. (1972). Prostaglandin generation maintains the smooth muscle tone of the rabbit jejunum. Br. J. Pharmac. 44, 328P.