University of Wi&consin, Madi8on, Wi8conein 53706, U.S.A.

Transcription

1 J. PhyioL (1977), 272, pp With 5 text-figure Prite4 in Grea Brtain ANTAGONISM BY INDOMETHACIN OF NEUROGENIC HYPERTHERMIA PRODUCED BY UNILATERAL PUNCTURE OF THE ANTERIOR HYPOTHALAMIC/PREOPTIC REGION BY T. A. RUDY, J. W. WILLIAMS AND T. L. YAKSH From the School of Pharmacy, Center for Health Sciences, University of Wi&consin, Madi8on, Wi8conein 53706, U.S.A. (Received 15 March 1977) SUMMARY 1. In unanaesthetized rats, restrained at an ambient temperature of 24 TC, the anterior hypothalamic/preoptic (AH/PO) region was lesioned unilaterally by acute mechanical puncture. 2. In control (no pre-treatment) rats, unilateral AR/PO puncture produced a neurogenic hyperthermia which began immediately, reached its peak magnitude (mean peak magnitude = ) within min and persisted usually for 8-16 hr. At defervescence, core temperature fell to a level near that of the pre-lesioning base line. 3. The prostaglandin synthesis inhibitor, indomethacin, administered I.P. at doses of 5 and 15 mg/kg 1 hr before puncture of the AR/PO region, attenuated the lesion-induced hyperthermia in a dose dependent fashion. The higher dose reduced peak magnitude by 80 % and the 6 hr Fever Index by 88 %. The vehicle used to dissolve the indomethacin (60 % DMSO/40 % saline) did not significantly attenuate the hyperthermia. 4. In rats that were hyperthermic after AR/PO damage, indomethacin (10-15 mg/kg i.p.) caused core temperature to fall promptly to near the prelesion base line. Reversal occurred whether the indomethacin was injected while core temperature was still rising or late in the plateau phase of the hyperthermia. 5. It is suggested that the neurogenic hyperthermia elicited by unilateral lesioning of the AH/PO region was mediated by prostaglandins released from injured tissue and possibly from extravasated blood. Evidence is cited indicating that the most likely sites of action of the released prostaglandins are the surviving portion of the AH/PO region on the punctured side and the intact contralateral AH/PO region.

2 722 T. A. RUDY, J. W. WILLIAMS AND T. L. YAKSH INTRODUCTION It is known that injury to the brain can sometimes cause an increase in body temperature. The term 'neurogenic' has been used to describe these fevers because they are thought to be caused by neuronal dysfunction rather than a bacterial or other exogenous pyrogen. One, of the best documented causes of neurogenic hyperthermia is injury to the rostral portion of the hypothalamus or the preoptic region. Hyperthermia has been produced by intentional lesioning of these regions in many animal species (cat: Teague & Ranson, 1936; Clark, Magoun & Ranson, 1939; dog: Keller & McClaskey, 1964; goat: Andersson, Gale, Hokfelt & Larsson, 1965; monkey: Ranson, Fisher & Ingram, 1937; rabbit: Barbour & Wing, 1913; rat: Anand & Brobeck, 1951; Gamble & Patton, 1953; Lipton, Dwyer & Fossler, 1974) and has been observed in man as a sequela of hypothalamic trauma produced by neurosurgical procedures, various disease states and head injury (Davison, 1940; Zimmerman, 1940; Sachs, 1945; Anderson & Haymaker, 1962). The typical neurogenic hyperthermia produced by rostral hypothalamic/preoptic trauma begins almost immediately and is frequently intense and short-lasting. A rise in body temperature to a single brief peak may be the only effect or it may be followed by persistent low grade hyperthermia, thermoregulatory instability or inability to maintain core temperature in the face of external heat or cold stress. The subject of this study is the invariable initial peak in core temperatures. Why core temperature increases rapidly after injury to the rostral hypothalamus or preoptic region is not known, but several speculative explanations have been offered. Some investigators (Clark et al. 1939; Squires & Jacobson, 1968) have suggested that destruction of tissue essential for heat dissipation is in part responsible for the effect, but all believed that the primary action of the injury was to produce a state of hyperactivity in neuronal systems controlling heat gain situated in the tissue adjacent to the destroyed area. Most often, this activation of perilesional tissue was attributed to an 'irritating' (Aronsohn & Sachs, 1885; White, 1890; Clark et al. 1939) or 'inflammatory' (Squires & Jacobson, 1968) effect of the injury. Although in no instance were these terms precisely defined, the impression given is that these authors related the hyperthermia to a non-specific local disturbance of function, engendered by processes such as mechanical damage, oedema, ischaemia or parenchymal haemorrhage. Barbour & Wing (1913), however, thought the peri-lesional stimulation was caused by specific pharmacological agents ('decomposition products') released from cells killed by the injury, and Andersson et al.

3 INDOMETHAACIN AND NEUROGENIC HYPERTHERMIA 723 (1965) believed that the injury released heat gain centres from a tonic inhibition normally emanating from the destroyed tissue. Prostaglandins of the E series are strongly pyrogenic when introduced into the ventricular cerebrospinal fluid or into the AH/PO region of a number of species (see Hellon, 1975, for references). Furthermore, in peripheral tissues (see Markelonis & Garbus, 1975, for references) and probably in brain (Dey, Feldberg, Gupta, Milton & Wendlandt, 1974; Wolfe & Mamer, 1975, Wolfe, Pappius & Marion, 1976), tissue injury stimulates the synthesis and release of prostaglandins. It seems possible, therefore, that prostaglandins released from injured tissue rather than disinhibition or a non-specific 'irritation' could be responsible for the hyperthermia caused by anterior hypothalamic or preoptic trauma. If this is true, a neurogenic hyperthermia so elicited should be prevented by an agent which inhibits the biosynthesis of prostaglandins. The present communication describes the effect of such a substance on the hyperthermia produced in rats by massive unilateral mechanical disruption of AH/PO region. The results strongly support the notion that prostaglandins mediate the hyperthermia which follows rostral hypothalamic or preoptic injury. METHODS Male Holtzman rats weighing g at surgery were used in these experiments. The rats were housed in individual cages at an ambient temperature of C in a room with controlled lighting (12 hr on-12 hr off). Food and water were available ad libitum. Each rat was anaesthetized with sodium pentobarbitone (30 mg/kg I.r.) and placed in a stereotaxic instrument set with the incisor bar elevated 5 mm above the interaural line (Pellegrino & Cushman, 1967). The skull was exposed and cleared of fascia and a small hole drilled at stereotaxic co-ordinates AP 7-6, L (Pellegrino & Cushman atlas, 1967). The dura was incised and a sterile, blunt tipped guide cannula was lowered into the brain so that its tip lay approximately 2 mm above the dorsal surface of the anterior commissure (about 5 mm below dura; HV + 2-0). The guide cannula was fashioned from a 9 mm length of 18 gauge stainless-steel tubing. The cannula was fixed to the skull using stainless-steel screws and cranioplastic cement and then occluded with a snugly fitting stainless steel stylet. The rats were replaced in their cages and permitted to recover for at least 14 days. Recovered rats were transferred to a temperature controlled room (24 ± 0-5 0C) and placed in individual hemicylindrical wire mesh cages which were sufficiently snug to prevent the animal's turning around. Core temperature was detected by a thermistor probe inserted 7-8 cm beyond the anus and taped to the base of the tail. Permanent records of core temperature were obtained using a bridge circuit and a multichannel recording potentiometer. After the colonic probe had been inserted, the rats were left undisturbed until core temperature had become stable (usually 1-2 hr). If the animal was destined to receive a drug or vehicle injection, the restraint cage cover was then briefly lifted and the agent quickly injected i.p. Core temperature was monitored for an additional hour following the injection. At this time, a sliding port over the animal's head was opened

4 724 T. A. RUDY, J. W. WILLIAMS AND T. L. YAKSH and the stylet removed from the guide tube and replaced with a sterile stylet 6 mm longer than the guide. The lesioning stylet was gently pushed down through the guide to the base of the brain, producing an instantaneous destruction of the anterior hypothalamic and preoptic tissue lying below the guide tip. Temperature recording was continued after lesioning for 8-24 hr, depending on the duration of the hyperthermia elicited. The stylet was not removed until the time the rat was sacrificed for histological examination of the brain. Scale drawings illustrating in the frontal and sagittal planes the positions and relative sizes of the guide and fully inserted lesioning stylet are shown in Fig. 1. Fig. 1. Sagittal and frontal representations of the rat brain illustrating the positions and sizes of the guide cannula and fully inserted lesioning stylet. The drawings are approximately to scale. The forty-eight rats used in the study were divided by random assignment into four groups of twelve animals each. One group received no pre-treatment injection. The second group received indomethacin, 5 mg/kg I.P.; the third, indomethacin, 15 mg/kg i.p. and the last, an i.p. injection of the vehicle used to dissolve the indomethacin (60 % spectral grade dimethylsulphoxide and 40 % normal saline, v/v). In

5 INDOMETHACIN AND NEUROGENIC HYPERTHERMIA 725 addition, in a few experiments carried out using additional animals, indomethacin was injected at various times following placement of the lesion rather than as a pretreatment. All injections were made in a volume of 3 ml./kg. The changes in core temperature elicited by the lesioning process were quantified on the basis of the maximum increase in core temperature observed within the 6 hr period following esioning (AT0, in 'C) and on the basis of a 6 hr fever index (FI,, in T -hr). The latter was obtained by planimetric measurement of the area enclosed between the fever curve and the extrapolated base line temperature. For the calculation of both AT0 andfi,, base line temperature was taken as the colonic temperature which existed just before insertion of the lesioning sylet. The data were subjected to a one-way analysis of variance and the individual means compared at the 0 05 level of significance using the Newman-Keuls test (Winer, 1971). Each lesioned animal was killed within 48 hr after lesioning and its brain perfused with saline followed by 10% formalin. The fixed brains were sectioned at 60,sm and stained with either cresyl violet or a modified Kluver-Barrera (1953) stain. Using a microprojector, the sections were projected onto frontal plates of the rat brain taken from the atlas of Pellegrino & Cushman (1967), and outlines of the lesioned areas were sketched in. In addition, the individual lesions were examined microscopically for evidence of hemorrhage. RESULTS Effect of unilateral puncture of the AH/PO region in control rat8 In each of the twelve animals in the control (non-pre-treated) animals (and in more than fifty additional animals employed in preliminary and subsequent experiments), the lesioning process produced a hyperthermic response. The temperature increases recorded from three of the twelve control animals are depicted in Fig. 2. The mean fever magnitude values for the entire group are given in Table 1. The typical hyperthermia produced by puncture of the AH/PO region was a monophasic rise in temperature which began almost immediately (mean latency = < 2 min), reached its peak magnitude within mi and lasted 8-16 hr, after which time, core temperature returned to a level near the pre-lesioning base line. Occasionally, responses as short-lasting as 4 hr or longer than 24 hr were seen. Also, in a few instances the fever curve was double peaked or the hyperthermia was preceded by a brief hypothermia. All the animals in the control group in this study had simple monophasic responses. None of the animals died within 24 hr after lesioning. Immediately after lesioning of the AH/PO region, some control rats were restless or agitated. In those that became excited, the agitation was superseded within 15 min by sedation, a demeanour which was maintained throughout the remainder of the rising phase and during the plateau phase of the response. During this period, the rats assumed a hunched, sleep-like posture and generally kept their eyes closed. However, they could be roused by auditory or tactile stimulation. Since some animals were not

6 726 T. A. RUDY, J. W. WILLIAMS AND T. L. YAKSH agitated during the rising phase and all were calm during the plateau phase, the temperature increase cannot be attributed to an increase in motor activity. Post-mortem examination of the sectioned and stained brains of control animals revealed that the acute puncture had obliterated unilaterally much of the AH/PO region in each rat. The extent of the largest and smallest lesions in the control series is illustrated on the left sides of the frontal sections of the rat brain shown in Fig. 3. The typical lesion consisted of a sharply defined cylindrical space surrounded by a layer of fragmented or torn tissue of variable thickness. Evidence of significant haemorrhage into 0_1 U+3 C H r Fig. 2. Effect on colonic temperature of unilateral, damage to the AH/PQ region in three control rats;. The puncture lesion was made at time 0 hr. as indicated by the arrow. the lesion or adjacent tissue was found in only six of the twelve animals, although some parenchymal bleeding undoubtedly occurred in the remiiganimals. In one case (the large lesion shown in Fig. 3), blood had flowed up the outside of the guide cannula into the lateral ventricle, causing extensive destruction in the tissue superjacent to the acute penetration. In every animal the anterior hypothalamic area, medial preoptic area, lateral preoptic area, suprachiasmatic nucleus, optic chiasm and supraoptic nucleus were damaged extensively. Frequently involved were the diagonal band of Broca, lateral paraolfactory nucleus and the ventral aspects of the medial and lateral septal nuclei. The ependyma of the third ventricle was frequently torn. Occasionally damaged were portions of the caudate nucleus, the dorsal portions of the medial and lateral septal nuclei, the fornix columns and the paraventricular nucleus. The lesions never damaged the ventromedial or more caudal hypothalamic nuclei. In

7 INDOMETHACIN AND NEUROGENIC HYPERTHERMIA 727 addition to the structures destroyed acutely, placement of the guide cannula had in every case destroyed superjacent structures (portions of the cyngulate and frontal cortex, corpus callosum, medium part of the caudate nucleus and much of the medial and lateral septal nuclei) ' Fig. 3. Frontal sections of the rat brain illustrating the extent of the largest and smallest lesions in the control series (left sides of sections) and in the series of rats pre-treated with indomethacin, 15 mg/kg (right sides of sections). Hatched areas = largest lesion. Stippled areas = smallest lesion. Boldface numbers indicate the AP plane of each section. The effect of indomethacin on hyperthermia elicited by unilateral puncture of the AH/PO region In Table 1 are summarized the hyperthermia magnitude data for the unpre-treated rats and those injected before lesioning with either indomethacin or its vehicle. The vehicle did not significantly affect the lesioninduced hyperthermia, whereas both doses of indomethacin significantly reduced both AT, and FI6. The 5 mg/kg dose reduced AT, by 39% and FI6 by 50 %, and the 15 mg/kg dose reduced ATc and FI6 by 80 and 88 %,

8 728 T. A. RUDY, J. W. WILLIAMS AND T. L. YAKSH respectively. The difference in the anti-hyperthermic effect of the two indomethacin doses was statistically significant. Presented in Fig. 4 are the temperature traces obtained from three rats pre-treated with indomethacin, 15 mg/kg. In each of these animals, the lesioning process elicited only a small temperature increase. The most common effect of indomethacin pre-treatment was simply to attentuate TABLE 1. The magnitude of neurogenic hyperthermia elicited by unilateral damage to the AH/PO region in control rats and in rats pre-treated with indomethacin or its vehicle (DMSO/saline). Pretreatments were administered ii'. 1 hr before lesioning. n = number of rats in each treatment group. ATO = mean maximum increase in colonic temperature within 6 hr of lesioning. FI, = mean area under the fever curve for the first 6 hr after lesioning. Means in a given column which are not connected by a vertical line differ significantly (P < 0.05) from all other means in the column. In control (no pre-treatment) rats, the mean core temperature just before lesioning was C (range = 36* C). The mean base line temperature in each of the other groups was approximately 0 9 0C lower (see text) AT0( ± s.e. of FI,( S.E. of Pre-treatment n mean (0C)) mean (0C hr)) None DMSO/saline Indomethacin 5mg/kg ± Indomethacin 15 mg/kg ± ± 0-39 Q +2 a, -c Hr Fig. 4. Effect of colonic temperature of unilateral damage to the AH/PO region in three rats pre-treated with indomethacin, 15 mg/kg, i.p. The puncture lesion (P) was made at time 0 hr. Indomethacin (I) was administered 1 hr later. the magnitude of the lesion-induced hyperthermia, the latency remaining unaffected (illustrated by the interrupted line in Fig. 4). Occasionally, a delayed rise in temperature was seen (depicted by the dotted line in Fig. 4), but these rises were small in comparison to the hyperthermias experienced by animals not protected with indomethacin.

9 INDOMETHACIN AND NEUROGENIC HYPERTHERMIA 729 In eight experiments, we examined the ability of an injection of indpmethacin (10-15 mg/kg) to reverse an already established hyperthermia. Both the early and the late phases of hyperthermia were found to be susceptible to indomethacin antagonism (Fig. 5). In both situations, core temperature fell rapidly to the pre-lesioning base line after administration of the drug. The hyperthermia shown by the interrupted line in Fig. 5 is one of the longest we have observed and is additionally unusual in that it is double-peaked. Both the low dose and the high dose of indomethacin and the indomethacin vehicle produced a decrease in body temperature in some rats. There was no significant difference in the hypothermic effect of the three treatments (mean fall in core temperature + S.E. of mean: indomethacin, 15 mg/kg = C; indomethacin, 5 mg/kg = , indomethacin vehicle = C). The magnitude of the fall varied considerably among animals and correlated poorly with the ability of a treatment to prevent lesion-induced hyperthermia (see Fig. 4) ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~- C) E +2 _ C I 0) m O~ _ I s II I I ", I ~~P O Hr Fig. 5. Reversal of established neurogenic hyperthermia by indomethacin in two rats. Hyperthermia was initiated by puncturing the AH/PO region unilaterally (P) at time 0 hr. In one rat, indomethacin, 15 mg/kg i.p., was injected while body temperature was still increasing. In the other rat, indomethacin, 10 mg/kg i.p., was injected late in the plateau phase of the response. Note the discontinuity in the abscissa. Post-mortem examination of the brains of lesioned rats pre-treated with indomethacin or its vehicle revealed no major differences in lesion size or position in comparison to the unpretreated rats. The largest and smallest lesions in the group of rats pretreated with indomethacin 15 mg/kg are illustrated on the right sides of the frontal sections shown in Fig. 3. About one third of the lesions in each group of treated rats showed evidence of significant haemorrhage into the lesioned area.

10 730 T. A. RUDY, J. W. WILLIAMS AND T. L. YAKSH DISCUSSION Massive unilateral mechanical destruction of the AH/PO region in control rats and in rats pre-treated with indomethacin vehicle produced an immediate sharp increase in core temperature which usually persisted for no more than 16 hr. Indomethacin, administered i.p. 1 hr before lesioning, attenuated the hyperthermia in a dose dependent fashion. Some of the rats pre-treated with indomethacin experienced a fall in core temperature before lesioning. However, it is not probable that the hypothermic action of indomethacin was in any appreciable extent responsible for indomethacin's ability to antagonize trauma-induced hyperthermia. Indomethacin effectively antagonized the fever normally produced by AH/PO puncture in animals in which it did not cause hypothermia. Moreover, the degree of hypothermia produced by the higher dose of indomethacin was no greater than that produced by the lower dose, whereas the higher dose was considerably more effective in preventing hyperthermia. In addition, the vehicle for indomethacin produced a fall in core temperature equal to that produced by both doses of indomethacin but had no significant anti-hyperthermic effect. Indomethacin is a potent inhibitor of prostaglandins biosynthesis in peripheral tissues and in brain (Flower, 1974; Wolfe et al. 1976). In incubated slices of rat cerebral cortex, its ID50 concentration for PGE2 and PGFb synthesis was only 0-28 FM, 100 and 1000 times smaller than those of aspirin and paracetamol, respectively (Wolfe et al. 1976). In many peripheral tissues, prostaglandins synthesis and release can be elicited by stimuli which can be considered injurious, including mechanical disturbance, thermal injury, ischemia and chemical assult (Markelonis & Garbus, 1975). There is evidence that brain tissue also responds to injurious stimuli by releasing prostaglandins. Elevated levels of prostaglandins have been detected in the cerebrospinal fluid of humans suffering from various neuropathologies and after neurosurgical procedures (Wolfe & Mamer, 1975). In the cat, the injection of endotoxin (Feldberg & Gupta,1973) and perfusion of the cerebroventricular system with an artificial cerebrospinal fluid (Dey et al. 1974) caused the concentration of prostaglandins in the cerebrospinal fluid to increase. Quick frozen rat cerebral hemispheres contained little or no prostaglandin, but immediately after slicing of the tissue, approximately 86 ng/g of PGE2a and 41 ng/g of PGE2 were present (Wolfe et al. 1976). Upon incubation of the slices in Ringer-bicarbonateglucose solution, they continued to release prostaglandins for more than an hour, ultimately yielding more than 200 ng/g of PGE2 and more than 800 ng/g of PGF2a. In most species thus far examined, prostaglandins of the E series have

11 INDOMETHACIN AND NEUROGENIC HYPERTHERMIA 731 proved to be extremely potent pyrogens when injected into the ventricular system or into the parenchyma of the AH/PO region (Hellon, 1975). In the. rat, as little as 0*1 ng injected into the AH/PO region bilaterally caused significant hyperthermia (Veale & Cooper, 1974a). The hyperthermia elicited by an injection of PGE1 into the AH/PO region of rat (Veale & Cooper, 1974a), rabbit (Stitt, 1973) or monkey (Crawshaw & Stitt, 1975) began almost immediately and was fever-like in that the temperature elevation was brought about by the coordinated activity of several thermoregulatory effectors and its magnitude was not influenced significantly by variations in ambient temperature. In only two studies, the present one and that of Andersson et al. (1965), has core temperature been recorded continuously in unanaesthetized animals following lesioning of the AH/PO region. In both studies, body temperature began to increase immediately after lesioning. Andersson et al. (1965), found that hyperthermia produced by lesioning of the AH/PO region in the goat was associated with strong shivering, peripheral vasoconstriction, adrenomedullary activation and increased plasma protein bound iodine and glucose levels. Although the effect of varying ambient temperature on the magnitude of the response was not tested, the hyperthermia was clearly a consequence of coordinated effector activity. In the rats which form the basis for the present report, we did not ascertain whether the hyperthermia produced by hypothalamic trauma was a regulated, fever-like response. However, in contemporaneous experiments in other rats, the hyperthermia produced by unilateral puncture of theah/po regions was found to be accompanied by tail vasoconstriction and shivering-like activity and its magnitude was not enhanced or diminished by variations in ambient temperature (D. Ackerman and T. A. Rudy, unpublished results). In certain respects, therefore, the hyperthermias elicited by an injection of PGE, into the AH/PO region and by lesioning of this region are similar. In view of the preceding discussion it seems reasonable to suggest that the immediate hyperthermia produced by unilateral puncture of the AH/ PO region in the present study was caused by the local release of prostaglandins and that the antagonism of this effect by indomethacin was a consequence of inhibition of prostaglandins biosynthesis and release. Dey et al. (1974) have suggested that the delayed fever which sometimes follows perfusion of the cerebral ventricles with artificial cerebrospinal fluid is due to prostaglandins released by mild injury to the ventricular ependyma. They implied that the delay represented the time required for prostaglandin biosynthesis to become activated. The present results suggest that when the injurious stimulus is severe and made directly within a prostaglandin sensitive area, the release of prostaglandins and associated fever can begin immediately.

12 732 T. A. RUDY, J. W. WILLIAMS AND T. L. YAKSH Indomethacin is a potent antagonist of the pyrogenic action of leukocytic pyrogen (Clark & Cumby, 1975), a member of a group of endogenously produced pyrogenic proteins, hereafter referred to as 'EnP' (terminology of Hellon, 1975). Although much evidence suggests that the effects of these agents are mediated ultimately by prostaglandins (Hellon, 1975), it is also possible that indomethacin and other antipyretics may act as antagonists at EnP receptors (Clark & Coldwell, 1972; Clark & Cumby, 1975). Since EnP injected into the AH/PO region produces a hyperthermic response similar to that produced by PGE1 (Cooper, Cranston & Honour, 1967; Jackson, 1967; Rosendorff & Mooney, 1971; Veale & Cooper, 1974a), the possibility that the fevers we observed were mediated by EnP rather than prostaglandins must be considered. If EnP were involved in the genesis of lesion-induced hyperthermia, its most likely source would be blood, for there is no evidence that brain tissue contains or synthesizes EnP. The blood of afebrile animals contains little or no pre-formed EnP (Atkins & Bodel, 1974). Polymorphonuclear cells or monocytes present in extravasated blood or entering the lesioned area by diapedesis through the walls of capillaries in peri-lesional tissue could probably secrete EnP after becoming activated by phagocytosis. However, even already activated leukocytes require 1-2 hr before they can release EnP (Atkins & Bodel, 1974). It is not likely, therefore, that an EnP is responsible for the early component of the hyperthermia elicited by AH/PO puncture. On the other hand, we cannot discount the possibility that an EnP might contribute to the maintenance of an established hyperthermia or that it might be responsible for the occasionally observed second peak of hyperthermia. Possible sources of the prostaglandins involved in the generation of lesion-induced hyperthermia include brain tissue and blood. Evidence that injured brain can release prostaglandins has been cited previously. Blood contains little pre-formed prostaglandin (McCosh, Meyer & DuPont, 1976), but during the process of aggregation, blood platelets rapidly synthesize and release appreciable quantities of prostaglandins (Smith, Ingerman, Rocsis & Silver, 1973; Ferreira, Ubatuba & Vane, 1976). Furthermore, leukocytes, after they have been activated by phagocytosis, can secrete prostaglandin (Higgs,& Youlten, 1972; McCall & Youlten, 1973). Although further investigation will be required, we tentatively discount extravasated blood -as a major source of prostaglandins in the present experiments because the extent of intraparenchymal bleeding correlated poorly with the magnitude of the hyperthermia elicited by the lesions. The anatomical site of action of prostaglandins released by the lesioning process is probably the surviving portion of the ipsilateral AH/PO region and/or the contralateral intact AH/PO region. Microinjection mapping

13 INDOMETHACIN AND NEUROGENIC HYPERTHERMIA 733 studies of the rat brain have failed to reveal any locus other than the AsH/ PO region which can mediate a prostaglandin-induced hyperthermia (Lipton, Welch & Clark, 1973; Veale & Cooper, 1975; Williams, Rudy, Yaksh & Viswanathan, 1977). That prostaglandins injected into the hypothalamic parenchyma can diffuse into the ventricular cerebrospinal fluid has been demonstrated (Veale & Cooper, 1974b). Thus, it seems likely that prostaglandins released by the injured half of the AH/PO region could reach the contralateral half even should the third ventricular ependyma remain intact. The immediate rise in core temperature produced by unilateral mechanical destruction of the AH/PO region in the rat was relatively short-lasting. Defervescence was probably not due to pharmacological desensitization of prostaglandin receptors, as Feldberg & Saxena (1971) found that a continuous intraventricular infusion of PGE, in the cat produced a sustained hyperthermia. In view of the accessibility of the contralateral, intact AH/PO region to released prostaglandin, expansion of the lesion so that all prostaglandin responsive tissue was ultimately destroyed also seems improbable. A more likely explanation is that prostaglandin secretion by traumatized cells is self-limiting, either because the cell dies or because it recovers from injury. Possibly, both events contribute to defervescence. The results of this study provide support for the concept (see Introduction) that neurogenic hyperthermia is in large measure due to activation of surviving peri-lesional tissue. In particular, our findings substantiate Barbour and Wing's early suggestion (1913) that this stimulation is mediated by chemical agents released by the injured tissue. The data argue against non-specific irritation of peri-lesional tissue or neuronal disinhibition as mechanisms responsible for the hyperthermia produced by our technique. In addition, the results suggest that destruction of tissue critical to proper heat dissipation probably did not contribute to the hyperthermia. In the first place, it is well known that the diencephalic thermoregulatory pathways are bilaterally redundant, and unilateral lesions are therefore not likely to produce passive defects in temperature control. Moreover, that thermolytic mechanisms were intact in the lesioned, hyperthermic animals is suggested by the ability of indomethacin to produce rapid defervescence and by the results of ancillary experiments indicating that the hyperthermia is a regulated response. Although the hyperpyrexias reported by Barbour & Wing (1913) and in a large number of even earlier papers (see Barbour & Wing, 1913, and Kornblum, 1925, for references) were produced by unilateral lesions, bilateral lesions of the AH/PO region were responsible for many of the hyperthermic episodes reported by authors cited in the Introduction. The partial or complete destruction of both halves of the AH/PO region

14 734 T. A. RUDY, J. W. WILLIAMS AND T. L. YAKSH engendered by bilateral lesioning makes an explanation based on the disinhibition of thermogenesis and/or the crippling of thermolytic mechanisms more tenable. On the other hand, neurogenic fevers elicited by bilateral damage, like those produced by unilateral damage, are usually transient. In addition, bilateral lesions resulting from slow destruction of tissue do not cause hyperthermia (Anderson & Haymaker, 1962; Andersson et al. 1965). These characteristics are difficult to reconcile with a hypothesis which imputes the hyperthermic effect of bilateral damage to a loss of function of the lesioned tissue, with consequent disinhibition of thermogenesis and/or failure of thermolysis. They are readily explained however, if it is assumed that prostaglandins mediate the hyperthermia. Prostaglandin release from injured tissue could well be transient videe 8upra), and it is not improbable that slow destruction of tissue might release prostaglandins in amounts too small to exert any hyperthermic effect. However, until further experiments are carried out, the possibility that unilateral and bilateral lesions of the AH/PO region produce hyperthermia by different mechanisms cannot be ruled out. This work was supported by United States Office of Naval Research Contract N C A preliminary report of these findings appeared in Neuroaci. Ab8. 2, 731 (1976). The indomethacin was kindly supplied by Merck Sharp and Dohme. REFERENCES ANAND, B. K. & BROBECK, J. R. (1951). Hypothalamic control of food intake in rats and cats. Yale J. biol. Med. 24, ANDERSON, E. & HAYMAKER, W. (1962). Disorders of the hypothalamus and pituitary gland. In Clinical Neurology, vol. 3, 2nd edn., ed. BAKER, A. B., pp New York: Hoeber-Harper. ANDERSSON, B., GALE, C. C., HOKFELT, B. & LAEssoN, B. (1965). Acute and chronic effects of preoptic lesions. Acta physiol. 8cand. 65, ARONSOHN, E. & SACHS, J. (1885). Die beziehungen des gehirns zur korperwarme und zum fieber. Pfluger8 Arch. ge8. Physiol. 37, ATKINS, E. & BODEL, P. (1974). Fever. In The Inflammatory Process, vol. 3, 2nd edn. ed. SWEIFACH, B. W., GRANT, L. & MCCLUSKEY, R. T., pp New York: Academic Press. BARBOUR, H. G. & WING, E. S. (1913). The direct application of drugs to the temperature centers. J. Pharmac. exp. Ther. 5, CLARK, G., MAGOUN, H. W. & RANSON, S. W. (1939). Hypothalamic regulation of body temperature. J. Neurophy8iol. 2, CLARK, W. G. & CoLDwiELL, B. A. (1972). Competitive antagonism of leukocytic pyrogen by sodium salicylate and acetaminiophen. Proc. Soc. exp. Biol. Med. 141, CLARK, W. G. & CumnY, H. R. (1975). The antipyretic effect of indomethacin. J. Phygiol. 248, COOPER, K. E., CRANSTON, W. I. & HONOUR, A. J. (1967). Observations on the site and mode of action of pyrogens in the rabbit brain. J. Phy8iol. 191, CRAWSHAW, L. I. & STITT, J. T. (1975). Behavioural and autonomic induction of prostaglandin E1 fever in squirrel monkeys. J. Phygiol. 244,

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