425

J. Phypiol. (1975), 249, pp. 425-434 With 5 text-ftgure8 Printed in Great Britain

IS BRAIN PROSTAGLANDIN SYNTHESIS INVOLVED IN RESPONSES TO COLD?

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

(Received 24 January 1975) SUMMARY

1. Experiments with rats have suggested that prostaglandin synthesis in the c.N.s. may mediate thermoregulatory reactions to cold. This possibility was investigated in cats using two types of experiment. 2. In one series of experiments, c.s.f. collected from the cisterna magna of conscious cats exposed to a cold and a hot environment was assayed for prostaglandin-like activity. During cold exposure there was a slight increase in activity which persisted after return to neutral ambient temperature. There was no correlation between prostaglandin-like activity and rectal temperature. During the heat exposure there was no demonstrable change in activity. 3. In the second series, conscious cats were exposed to cold conditions and given intravenous injections of salicylate, paracetamol, or indomethacin, all of which inhibit prostaglandin synthesis. Indomethacin caused a small, statistically significant fall in rectal temperature. Neither salicylate nor paracetamol caused any significant change in rectal temperature. 4. The results do not support a role for c.N.s. prostaglandin synthesis in thermoregulatory reactions to cold in cats. INTRODUCTION

Antipyretic drugs are generally considered to have no effect on the body temperature of afebrile animals. However, there are reports that antipyretics, when administered parenterally in cold or thermoneutral environments, have lowered the deep body temperatures of afebrile rats (Satinoff, 1972; Francesconi & Mager, 1974; Feldberg & Saxena, 1975) and cats (Milton & Wendlandt, 1968; Clark, 1970; Wendlandt, 1972; Milton, 1973;

426 W. I. CRANSTON, R. F. HELLON AND D. MITCHELL Dey, Feldberg, Gupta, Milton & Wendlandt, 1974). One of the actions of antipyretic drugs is to inhibit prostaglandin synthesis (Vane, 1971; Flower, 1974). Satinoff (1972) has therefore suggested that antipyretics exert their hypothermic effect on afebrile animals by blocking central nervous system (c.N.s.) prostaglandin synthesis which might normally be augmented as part of the physiological response to cold. This suggestion is supported circumstantially by several independent observations. Injections of the E series prostaglandins into the cerebral ventricles or the anterior hypothalamus in animals of several species cause a sharp rise in body temperature (Milton & Wendlandt, 1970; Feldberg & Saxena, 1971; Stitt, 1973). During fever, which may be associated with physiological reactions similar to those seen during cold stress, there is an increase in the concentration of prostaglandin in the cisternal cerebrospinal fluid (c.s.f.) (Feldberg & Gupta, 1973; Philipp-Dormston & Siegert, 1974). Antipyretics reduce fever and c.s.f. prostaglandin level (Feldberg, Gupta, Milton & Wendlandt, 1973; Dey et al. 1974). In the present experiments we attempted to find evidence to support a role for prostaglandin synthesis and release in the c.N.s. of cats exposed to cold. First, since little quantitative evidence is available on the hypothermic action of antipyretics in cats, we administered salicylate, paracetamol and indomethacin intravenously to cats exposed to a standard cold stress, and recorded rectal temperature. Secondly, we collected c.s.f. from the cisterna magna of cats before, during, and after cold exposure, and measured its prostaglandin-like activity; this activity was also measured in c.s.f. obtained from some of the same cats exposed to heat. METHODS Animals. Ten female cats weighing between 2-5 and 3-5 kg were used. Under pentobarbitone anaesthesia (36 mg/kg) a stainless-steel guide tube was implanted with its tip just above the atlanto-occipital membrane (Feldberg et al. 1973). Strict aseptic precautions were observed. At least seven days elapsed before any experiments were performed and subsequently between any two experiments on the same animal. In all experiments the cats were conscious and mobile. Climatic exposure. The cats were placed singly in a small wind tunnel (Hellon, 1970) which allowed the animal a space measuring 500 x 250 x 350 mm. Conditioned air passed through the tunnel at a speed of 05 m/sec. Air temperatures were maintained between 6 and 90 C for the cold exposures and between 39 and 41° C for the hot exposures. Before and after these exposures the animals were kept in an environment of between 21 and 230 C. Procedures. In all experiments, deep body temperature was recorded at 5 min intervals using an indwelling rectal thermistor. Before the animals were placed in the wind tunnel, a fine nylon cannula was introduced into a cephalic vein and taped securely to the forelimb. Drugs could then be administered intravenously without disturbing the animal. Before and after all injections the cannulae were filled with a

427 PROSTAGLANDINS AND COLD EXPOSURE solution containing heparin (50 i.u./ml.). Both rectal thermistors and

0-9 % NaCl intravenous cannulae were well tolerated by the cats. The antipyretics were administered after the animals had been exposed to cold for 1 hr. Exposure and recording continued for a further hour. As a control procedure, the vehicles in which the antipyretics had been dissolved were administered to the same cats in other experiments in the same environmental conditions. In the experiments concerning indomethacin, the volume of vehicle used for control purposes was the same as that used to carry the drug. In those concerning salicylate and paracetamol the vehicle was saline, and a single series of control experiments was conducted, the volume injected being 3-5 ml./kg. Four cats were also exposed to the same cold environment without any injection. The collection of c.s.f. was undertaken in separate experiments. Before the cat was exposed to cold a sharpened sterilized steel cannula (24 gauge) was lowered into the implanted guide tube (Feldberg & Gupta, 1973). When the membrane over the cisterna had been pierced and c.s.f. flowed freely, the cannula was fixed in position. Sometimes slight adjustments of the position were necessary during an experiment to provide free flow. Samples of c.s.f. were drawn off through a syphon tube attached to the top of the cannula. Four samples were taken in each experiment: the first before exposure, the second after 1 hr of exposure, the third after 2 hr, and the fourth after 1 hr of recovery in the neutral environment. The volume of each sample was 0-6 ml. Eight cats were exposed to the cold environment, and three of them to the hot environment. The c.s.f. was assayed for prostaglandin-like activity in the rat fundus strip preparation (Feldbergetal. 1973). Prostaglandin E2was used as the standard. The method of measurement had a threshold of about 1 ng/ml. Any sample with activity below threshold was assigned the maximum subthreshold concentration possible, namely 1 ng/ml. Drug. Sodium salicylate was dissolved in pyrogen-free 09 % NaCl solution ('saline') in a concentration of 12-5 mg/ml. The dose administered was 120 mg/kg body wt. Paracetamol (4-acetamidophenol, B.P.) was also dissolved in saline. The concentration was 14 mg/ml. and the dose 50 mg/kg. Indomethacin (Merck, Sharp & Dohme) was made up at a concentration of 10 mg/ml. in a vehicle consisting of polyethylene glycol 200 (2 parts) and saline (1 part). A dose of 2 mg/kg was used. RESULTS

None of the cats was febrile at the start of any experiment. The mean values and ranges of rectal temperature at zero time for the various experimental and control treatments are given in Table 1. None of the means exceeded 39 00 C and the highest individual value was 39.3 C. During exposure to cold the cats exhibited typical cold defence mechanisms. They adopted a huddled posture and showed pilo-erection. In most cases they shivered vigorously. These reactions were well established within the first hour of exposure. Antipyretic drugs. None of the antipyretic drugs, injected intravenously, had any effect on the visible behaviour of the animals during cold exposure. Following the injection of salicylate 120 mg/kg there was a slow rise in

428 W. I. CRANSTON, R. F. HELLON AND D. MITCHELL TABLE 1. Rectal temperatures of cats before injection (antipyretic injection experiments) and before climatic exposure (c.s.f. collection experiments)

No. of

Type of experiment Antipyretic injection: None Saline Salicylate Paracetamol Indomethacin vehicle Indomethacin C.s.f. collection: Cold Heat

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,

cats

,

Rectal temperature (0 C) A Mean S.E. Range

4

38-87

0 16

5 6 6 6 6

38*20 38-26 38-64 38-12 38-20

0*24

8 3

39 00 38-28

384-39-0 37*4-38*9 38-1-38-7 383-39-0 37*5-38*7 38-038-4

0 10 0-12 0.19 0-08

0*08

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10 20 30 50 60 40 Time from injection (min) Fig. 1. Changes of rectal temperature (Tre) (mean ± s.E.) in cats exposed to cold and given intravenous injections of salicylate (@ *, six animals) and saline (O --- 02 five animals) after 60 min of exposure (zero time). 0

rectal temperature in all the cats. Fig. 1 shows the mean values for the group of six cats: the rise amounted to an average of 0-32 + 0.060 C over the last 15 min of exposure. In the control experiments, saline 3-5 ml./kg was injected into five of the cats. The mean change in rectal temperature (Fig. 1) showed a slow biphasic rise reaching 0-21 + 0.06° C over the final 15 min. The differences between the two curves were significant at the 5 % level 25 and 40 min after injection (t = 2-57 and 2-30 respectively), but not at any other time. The average values over the last 15 min were not significantly different. The effect on rectal temperature of exposing four of the cats to the same cold environment without any injection is shown in Fig. 2. The mean changes in rectal temperature after zero time were less than 0.10 C. In other words, the cats normally demonstrated effective regulation of body

429 PROSTAGLANDINS AND COLD EXPOSURE temperature in this cold environment. Comparison of the mean changes in rectal temperature after saline injection and after no injection revealed a significant (P < 0.05) difference only at 55 min after time zero (t= 268). The average change over the final 15 min following injection of saline was not significantly different from the average change in cats receiving no

injection.

60 30 40 10 20 50 Time from injection (min) Fig. 2. Changes of rectal temperature (Tre) (mean ± s.E.) in cats exposed to *, cold from -60 min and given intravenous injections of saline (0 five animals) at zero time or no treatment (0 - - - 0, four animals). The saline curve is the same as that in Fig. 1. 0

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Fig. 3. Changes of rectal temperature (Tre) (mean + s.E.) in cats exposed to *, six animals) cold and given intravenous injections of paracetamol (0 and saline (0 0O five animals) at zero time. The saline curve is the same as that in Figs. 1 and 2. ---

When paracetamol 50 mg/kg was injected into six cats under cold conditions, there were negligible changes in their rectal temperatures. The mean results are shown in Fig. 3. The responses following paracetamol were not significantly different from those following saline injection at any time. Indomethacin injection was followed by a mean fall in rectal temperature

430 W. I. CRANSTON, R. F. HELLON AND D. MITCHELL amounting to 0X29 + 0.140 C over the final 15 min of exposure. The fall was complete in 20 min (Fig. 4) and temperature remained steady for the remainder of the cold exposure. Also shown in Fig. 4 are the results of the control experiments in the same cats when the appropriate volume of vehicle was injected. There was then a slow rise in rectal temperature similar to that seen after saline, reaching 0 13 + 0.060 C over the final 15 min. The difference between the mean rectal temperature change following indomethacin and that following the vehicle was significant (P < 0.05) 10 min after the injection and throughout the rest of the exposure. The average difference over the final 15 min of exposure, namely 0-42 + 0.150 C, was also significant at the 5 % level. 0-3 _

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Fig. 4. Changes of rectal temperature (Tne) (mean ± s.E.) in cats exposed to cold and given intravenous injections of indomethacin (@ *, six animals) and indomethacin vehicle (0 --- 0O six animals) at zero time.

Prostaglandin activity. The results in Fig. 5 show that the cats again exhibited effective thermoregulation during the cold exposures in which c.s.f. was collected: the mean change in rectal temperature was not significant. Prostaglandin-like activity in the c.s.f. was initially below the threshold of the assay in all animals. In some animals activity remained below threshold, but mean activity for the group rose to 1-6 + 0'3 ng/ml and 2-5 + 0.5 ng/ml. after 1 and 2 hr of exposure to cold. One hour after return to a neutral ambient temperature, the prostaglandin activity fell to 1*8 + 0*2 ng/ml. The activity of the second sample taken in the cold was significantly different from pre-exposure activity (paired difference test t = 2-83, P < 0.05), but the activity of the first sample was not. Neither

431 PROSTAGLANDINS AND COLD EXPOSURE sample had an activity significantly different from the activity of the sample taken after the recovery. The foregoing calculations were based on the assignment of the threshold activity of the assay, namely 1 ng/ml., to all subthreshold samples. This assumption will affect mean values when some samples in the group are subthreshold, and therefore might bias the statistical comparisons. To test I

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60 90 120, 180 Time (min) Fig. 5. Changes in rectal temperature (Tr,) (mean + s.E.) and c.s.f. prostaglandin activity (mean ± s.E.) in eight cats exposed to neutral and cold environments. The horizontal bar indicates the period of cold exposure. C.s.f. samples with subthreshold activity were assigned the value 1 ng/ml. 0

30

this possibility the calculations were repeated with the same subthreshold samples assigned zero activity. The mean levels of activity then became 1.1 + 0.4 ng/ml. after 1 hr of exposure, 2-0 + 0-6 ng/ml. after 2 hr and 1-6 + 0*4 ng/ml. after recovery. Again, the activity of the second sample taken in the cold was significantly different from pre-exposure activity (paired difference test t = 2-71, P < 0.05), but the activity of the first was not, and neither activity differed from the activity of the sample taken after recovery. Hence the numerical value assigned to subthreshold activity does not affect the conclusions drawn. In the cats exposed to high ambient temperatures, mean rectal temperature rose by about 0 5 + 0.40 C. C.s.f. prostaglandin activities were below threshold before, during, and after the exposures. Dey et al. (1974) had speculated that the range of rectal temperatures observed in cats in the laboratory may be due to varying rates of synthesis of prostaglandin. Our results did not support this idea. The correlation between

432 W. I. CRANSTON, R. F. HELLON AND D. MITCHELL rectal temperature observed during the standard cold exposures and the concentration of prostaglandin in cisternal c.s.f. was far removed from statistical significance (product-moment correlation coefficient = 0-136; P > 0.1). DISCUSSION

None of the cats used in our experiments showed any rectal temperatures which approached febrile levels. It is therefore reasonable to assume that any drug effects which were observed were due to the effects of the drugs either on peripheral tissues or upon the normal C.N.S. processes mediating temperature regulation. In the cats given salicylate in a cold environment, there was no evidence of a fall in temperature like that reported by Satinoff (1972) and others for rats. The action of salicylate in cats exposed to cold is therefore the same as its action in cats exposed to thermoneutral environments (Clark, 1970) and also the same as its action in thermoneutral environments in rabbits (Cranston, Luff, Rawlins & Rosendorff, 1970; Cranston, Hellon, Luff, Rawlins & Rosendorff, 1970) and man (Rosendorff & Cranston, 1968). The dose of salicylate given in our experiments was of the same order as that used by Satinoff (1972) and others, and it is difficult to explain the difference between the effects observed in rats and cats. The different routes of administration (intraperitoneal in rats and intravenous in the present experiments) might be a factor; intraperitoneal injection of salicylate has been reported to be very irritant (Milton, 1973). Paracetamol had no effect on the temperature of our cats during exposure to cold. There have been occasional reports of paracetamol causing a fall in temperature in afebrile cats exposed to thermoneutral environments (Milton & Wendlandt, 1968; Clark, 1970; Wendlandt, 1972; Milton, 1973; Dey et al. 1974), but in most cases the effect was seen in cats whose starting temperatures were appreciably higher than the temperatures of our cats. The drug may therefore have been acting to reduce a slight fever, a suggestion already made by Wendlandt (1972). The effect of indomethacin on cats was to produce a small but significant reduction in rectal temperature. It seems unlikely that inhibition of prostaglandin synthesis was involved: paracetamol, which is also a potent inhibitor of prostaglandin synthesis in the brain (Flower, 1974), had no effect. We support the contention of Dey et al. (1974) that the hypothermic action of indomethacin in afebrile animals is unrelated to brain prostaglandin. In summary, therefore, of the antipyretic drugs tested, only indomethacin produced a significant fall in rectal temperature of cats exposed to cold. Though significant, the fall was meagre, having nothing like the

43 PIOSTAGLANDINS AND COLD EXPOSURE 433 magnitude of the fall seen in rectal temperature when indomethacin is administered to a febrile cat (Feldberg et at. 1973; Dey et al. 1974). The failure of salicylate or paracetamol to cause a fall in temperature suggests that augmented prostaglandin synthesis does not play a major role in cold defence. This suggestion is supported by the analyses of cisternal c.s.f. for prostaglandin-like activity. There was a statistically significant increase in prostaglandin-like activity after two but not after one hour of cold exposure. The activity attained was much lower than that seen during fever (Feldberg et al. 1973), and the slight elevation was maintained after one hour of recovery. We agree with Wendlandt (1972) that the evidence from c.s.f. prostaglandin activity does not suggest augmented synthesis or release of prostaglandin during either cold or heat exposure. We are grateful to Professor W. Feldberg, F.R.S. for his encouragement and advice, to M. Dashwood for performing the prostaglandin assays, to Margaret Tester for assistance with the experiments, to G. Duff for his helpful comments on the manuscript, Dr J. E. Pike of the Upjohn Company for providing us with samples of prostaglandin, and to the Pharmacy, St Thomas's Hospital, for preparing the antipyretic

drugs. REFERENCENS CLARK, W. G. (1970). The antipyretic effects of acetaminophen and sodium salicy. late on endotoxin-induced fevers in cats. J. Pharmac. exp. Ther. 175, 469-475. CRANSTON, W. I., HELLON, R. F., LUFF, R. H., RAwuxNs, M. D. & ROSENDORFF, C. (1970). Observations on the mechanism of salicylate-induced antipyresis. J. Physiol. 210, 593-600. 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. 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. & GUPTA, K. P. (1973). Pyrogen fever and prostaglandin-like activity in cerebrospinal fluid. J. Physiol. 228, 41-53. FELDBERG, W., GUPTA, K. P., MILTON, A. S. & WENDLANDMT, S. (1973). Effect of pyrogen and antipyretics on prostaglandin activity in cisternal CSF of unanaesthetized cats. J. Physiol. 234, 279-303. FELDBERG, W. & SAXENA, P. N. (1971). Further studies on prostaglandin El fever in cats. J. Physiol. 219, 739-745. FELDBERG, W. & SAXENA, P. N. (1975). Effects on body temperature of rats produced by prostaglandins, endotoxin, lipid A and antipyretics. J. Physiol. 245, 101P. FLOWER, R. J. (1974). Drugs which inhibit prostaglandin biosynthesis. Pharmac. Rev. 26, 33-67. FRANCEscoNI, R. & MAGER, M. (1974). Tryptophan and salicylate hypothermia. Fedrn Proc. 33. 408. HELLON, R. F. (1970). The stimulation of hypothalamic neurones by changes in ambient temperature. Pfluigers Arch. ges. Phyaiol. 321, 56-66.

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W. 1. CRANSTON, R. F. HELLON AND D. MITCHELL

MILTON, A. S. (1973). Prostaglandin E1 and endotoxin fever, and the effects of aspirin, indomethacin, and 4-acetamidophenol. Adv. in Biosci. 9, 495-500. MILTON, A. S. & WENDLANDT, S. (1968). The effect of 4-acetamidophenol in reducing fever produced by the intracerebral injection of 5-hydroxytryptamine and pyrogen in the conscious cat. Br. J. Pharmac. Chemother. 34, 215-216P. MILTON, A. S. & WENDLANDT, S. (1970). A possible role for prostaglandin E1 as a modulator for temperature regulation in the central nervous system of the cat. J. Physiol. 207, 76-77P. PHILIPP-DORMSTON, W. K. & SIEGERT, R. (1974). Identification of prostaglandin E by radioimmunoassay in cerebrospinal fluid during endotoxin fever. Natutwisenschaften 61, 134-135. ROSENDORFF, C. & CRANSTON, W. I. (1968). Effects of salicylate on human temperature regulation. Clin. Sci. 35, 81-91. SATINOFF, E. (1972). Salicylate: action on normal body temperature in rats. Science, N.Y. 176, 532-533. STITT, 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. WENDLANDT, S. (1972). Some factors involved in the control of body temperature and the action of pyrogens and pharmacologically active substances in modifying temperature regulation. Ph.D. Thesis. University of London.

Note added in proof. Since this work was completed some comparable data on rabbits have been published (Pittman, Q. J., Veale, W. L. & Cooper, K. E. (1974). Proc. Soc. Neuroscience, 4, 375). The results confirm the absence of any noticeable effect of intravenous salicylate on thermoregulation in rabbits exposed to a temperature of 100C.