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Vagotomy attenuates the behavioural but not the pyrogenic effects of interleukin-1 in rats
Autonomic Neuroscience: Basic and Clinical 85 (2000) 127–132 www.elsevier.com / locate / autneu
Vagotomy attenuates the behavioural but not the pyrogenic effects of interleukin-1 in rats Giamal N. Luheshi a , *, Rose-Marie Bluthe´ b , David Rushforth a , Nicholas Mulcahy a , Jan-Pieter Konsman b , Michael Goldbach a , Robert Dantzer b a
School of Biological Sciences, University of Manchester, Manchester M13 9 PT, UK b ¨ , 33077 Bordeaux cedex, France INRA-INSERM U394, rue Camille Saint-Saens Received 4 February 2000; accepted 23 May 2000
Abstract Vagal afferent signals, have been implicated in cytokine mediated interactions between the periphery and the central nervous system. Studies in experimental animals have shown that cytokine induced activation of brain mediated responses to infection such as fever, sickness behaviour and pituitary-adrenal activation, are inhibited by subdiaphragmatic vagotomy. We have previously proposed that the peripheral signal to the brain in fever is of a humoral nature while others have suggested that either neural afferents or a mixture of both humoral and neural signals may be involved. The objective of the present study was to examine further the role of vagal transmission, in mediating the febrile response to a systemic injection of IL-1b in rats and to compare this with changes in social exploration behaviour. Intraperitoneal injection of IL-1b (1.0–30.0 mg / kg) inhibited social exploration in rats and this was attenuated in vagotomized animals. Injection of increasing concentrations of IL-1b (0.1–1.0 mg / rat) induced significant (P,0.001) increases in core body temperature. However, in contrast to effects on social exploration, the increase in temperature was not inhibited by vagotomy at any of the doses used. These observations demonstrate a dissociation between the two brain mediated events, one of which is dependent on the integrity of the vagus nerve (social exploration) while the other (fever) is apparently generated by different mechanisms which may include circulating pyrogens. 2000 Elsevier Science B.V. All rights reserved. Keywords: Fever; Cytokines; CNS; Social interaction
1. Introduction Cytokines such as interleukin (IL)-1b are key mediators of the interaction between the immune and central nervous system (CNS) (Hopkins and Rothwell, 1995). Systemic administration of IL-1b in experimental animals results in the same CNS mediated responses associated with sickness behaviour (Rothwell, 1991) which include fever and decrease in social interaction behaviour. The mechanism by which systemically produced large hydrophilic molecules such as IL-1b interact with the brain is controversial but a number of hypothesis have been proposed. These include an active transport mechanism (Banks et al., 1991), access through areas devoid of the blood brain barrier (BBB) such as organum vasculosum of the laminae *Corresponding author. Present address. Douglas Hospital Research Centre, McGill University, 6875 Boulevard LaSalle, Verdun (Quebec), Canada H4H 1R3. Tel.: 11-514-761-6131; fax: 11-514-762-3034. E-mail address: [email protected] (G.N. Luheshi).
terminalis (OVLT) (Blatteis, 1992), or action on receptors situated on endothelial cells lining cerebral blood vessels, resulting in the release of secondary mediators (Van Dam et al., 1996). More recently, the involvement of an afferent neuronal link from the periphery to the brain has gained prominence. A number of recent reports have demonstrated a role for the vagus nerve in transmitting cytokine signals to the brain to elicit an array of host defence responses. These responses, which included fever, were shown to be significantly inhibited in vagotomized (VGX) experimental animals injected peripherally with inflammatory stimuli including IL-1 [see (Watkins et al., 1995a; Maier et al., 1998) for extensive reviews of this area]. However other studies on fever have demonstrated the importance of mechanisms other than neuronal afferents. The most likely of these is the involvement of circulating pyrogens such as IL-6, which has been shown to increase dramatically in the circulation of febrile animals after treatment with, for example LPS (Luheshi et al., 1996; Miller et al., 1997).
1566-0702 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S1566-0702( 00 )00231-9
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It has been suggested that the attenuation of fever and other responses observed in VGX animals may be dependent on the dose of the inflammatory stimulus used (Gaykema et al., 1995; Hansen and Krueger, 1997). Hansen and Kruger (Hansen and Krueger, 1997) demonstrated that increasing the dose of i.p. IL-1b overrides the attenuation in brain temperature and non-rapid eye movement sleep (NREMS) observed in VGX animals. These observations suggested that a pathway other than the vagal afferent is involved in mediating the IL-1b signal from the periphery to the brain. Other workers (Romanovsky et al., 1997) have shown that in VGX animals injected with increasing concentrations of i.v. LPS only the monophasic fever resulting from a low dose of LPS was attenuated while the biphasic fever (in response to higher doses of LPS) remained unaffected by vagotomy. These workers concluded that vagal afferents might be important in mediating responses to minor concentrations of LPS, whereas the response to a higher dose may be mediated by non-vagal mechanisms. Other studies demonstrated that selective vagal rootlet deafferentation failed to completely block the suppression of food intake induced by either LPS or IL-1b administered systemically (Schwartz et al., 1997). Our own preliminary studies (Luheshi, 1998) indicated that fever was not attenuated in VGX animals following i.p. administration of a single dose of either LPS or IL-1b. Other workers (Caldwell et al., 1999a) arrived to a similar conclusion following burn injury in rats. It is obvious from the number of studies performed in this area that conflicting data exists. The majority of studies quite clearly suggest that the vagus plays an important part in mediating cytokine activated signals to the brain, however depending on the dose of the stimulus used or the parameter being measured, this part may not be exclusive and other mediators may be involved. The aim of the present investigation therefore, was to address some of these discrepancies, by (1) performing dose response studies using IL-b administered systemically (i.p.) to study the role of the vagus nerve in the development of fever; and (2) by using the same approach, compare the fever responses with those to changes in social behaviour, a parameter that has been consistently shown to be attenuated by vagal DEAFFRENTIATION.
2. Methods
2.1. Animals All experiments were performed on adult, male, Wistar rats (Charles River, France and Charles River, UK) of 225–250 g body weight. The animals were housed in a temperature controlled room (21.062.08C) artificially lit from 08:00 A.M. to 08:00 P.M., and were provided with food (Extralabo, Provins, France or Beekay international, UK) and water ad libitum. Juvenile male rats (21–35 days
of age) of the same strain served as stimulus animals for the behaviour studies.
2.2. Surgery Animals were food deprived for 24 h prior to surgery and randomly assigned to undergo either vagotomy or sham operation as previously described by Goldbach et al. (Goldbach et al., 1997) modified for the rat. Briefly, animals were anaesthetised with a mixture of ketamine and xylazine (61.0 and 9.0 mg kg 21 respectively), and the main ventral and dorsal trunks of the vagus nerve were transected immediately above the stomach. All connective tissue 2–3 cm between the gastric artery and the oesophagus was removed to ensure the transection of any accessory vagal branches. Sham animals underwent the same surgical procedure excluding transection of the nerve. All rats received a single intramuscular injection of AMOXCILLIN (75.0 mg kg 21 ) and were allowed to recover from surgery for 4 weeks before experimentation. The surgery was performed by the same investigator for both the fever and behaviour studies. Vagotomy was verified by isolectin I B4 (1 mg / ml phosphate buffered saline; L5391, Sigma) binding in the NTS. Most vagal afferents project to the NTS and isolectin I B4 has the peculiarity of binding to vagal endings in this region (Li et al., 1997). The distribution of isolectin I B4 in the NTS of sham operated rats was similar to that reported previously. High isolectin I B4 binding was found in the medial and commissural nuclei as well as in the NTS of shamoperated animals. After subdiaphragmatic vagotomy, isolectin I B4 binding in these structures was highly reduced (data not shown).
2.3. Experimental Hand-held, lightly-restrained rats, were injected i.p., (1.0 ml / kg) with rat recombinant IL-1b (biological activity: 317 IU / mg, Dr S. Poole, NIBSC, Potters Bar, UK) dissolved in pyrogen free saline containing 0.1% endotoxin-free bovine serum albumin (BSA, A-8806, Sigma, USA) and using a dose range of 0.1–1.0 mg / kg for fever studies and 1.0–30.0 mg / kg for behaviour (Anforth et al., 1998). Control animals received an equal volume of 0.9% sterile saline. Behaviour studies were conducted as a between–within comparison, the surgery (sham versus vagotomy) and dose (1.0, 3.0, 10.0 and 30.0 mg / kg) factors were considered as between factors; the treatment (IL-1b versus vehicle) was considered as a within factor.
2.3.1. Fever Core body temperature was measured continuously in conscious, undisturbed, individually housed animals (n5 10–15) by remote radio-telemetry, via battery-operated biotelemetry transmitters (Data Sciences, St. Paul, USA), previously implanted in the abdominal cavity during the
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vagotomy or sham procedures. The output frequency (Hz) of each transmitter was monitored by an antenna mounted in a receiver board situated beneath the cage of each animal, and channelled to a peripheral processor (Dataquest IV, Data Sciences). Frequencies were sampled at 10-min intervals and converted to degrees Celsius (8C). Animals received a single injection of IL-1b or vehicle at 10:00 A.M. and their temperatures were monitored for 7 h.
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sponded to the lowest dose of IL-1b (0.1 mg, Fig. 1a) while increasing the dose to 0.5 (Fig. 1b) and 1.0 mg / kg (Fig. 1c) resulted in a highly significant febrile response in both groups (P,0.001) of animals when compared to their vehicle treated controls. In both cases, a stress induced transient rise in temperature was followed by a sharp increase, which peaked at 2.5 h after which it declined
2.3.2. Social exploration IL-1b-induced sickness behaviour was assessed in sham (n524) and VGX (n524) rats by the reduction in duration of social investigation of a conspecific juvenile introduced momentarily into the home cage of the test animal at various times before and after i.p. injection of either IL-1b or vehicle. Social investigation was measured by the duration of ano-genital sniffing and close following of the juvenile during a 4-min session immediately before the injection and 2 and 4 h later. Different juveniles were presented on each occasion. 2.3.3. Statistical analysis Data from fever experiments are presented as means6S.E.M. Temperature data were analysed as area under the curve (AUC) and one-way ANOVA followed by Scheffe’s post-hoc test. In the behaviour studies in order to verify that surgery did not affect the behaviour of VGX animals compared with sham-operated animals, duration of social investigation (seconds) measured during the first session before injection of vehicle or IL-1b was submitted to a one-way ANOVA. The mean duration of social investigation over the 4 h period following injection of vehicle or IL-1b was expressed as percentage of baseline and submitted to a three-way ANOVA, doses of IL-1b and surgery as between factors and treatment as within factor. Post-hoc comparisons of individual group means were carried out by the protected least, significant difference test (LSD).
3. Results
3.1. Effect of subdiaphragmatic vagotomy on IL-1b induced fever The basal temperatures of animals used in these experiments ranged from 36.58C to 36.88C and were consistently lower in the VGX (36.560.18C) compared with the sham controls (36.860.18C, P,0.01, n536). The basal temperatures rose moderately in all rats at the beginning of each experiment, probably due to handling stress during injection. This initial rise in temperature normally persisted for 1 h, after which it returned to basal levels. Injection of IL-1b (0.1–1.0 mg / kg, i.p.) induced a dose dependent monophasic fever in both sham and VGX animals. Neither VGX, nor sham operated animals re-
Fig. 1. Effect of vagotomy on core body temperature: Changes in core body temperature of vagotomized and sham operated rats after i.p. injection of different doses of IL-1 (n510–15) or vehicle (1.0 ml / kg, n510–15) at time 0 h. Values are means6S.E.M (n510–15) and were analysed using area under the curve (AUC;8C / min).
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towards basal levels (Fig. 1b and c). There was no difference in the magnitude of the febrile response to IL-1b, exhibited by either VGX or the sham animals. However, VGX animals injected with 1.0 mg / kg IL-1b failed to sustain febrile response compared to counterpart sham controls (Fig. 1c). In this case the fever peaked at 2.5 h after which it declined more rapidly towards basal temperature when compared with the shams where the rise in temperature was maintained for up to 5 h.
3.2. Effect of subdiaphragmatic vagotomy on IL-1b induced depression in social exploration A one-way ANOVA on the duration of social investigation measured immediately prior to treatment did not reveal any difference between VGX and sham rats (mean duration of investigation: 127.965.0 s in sham vs. 137.264.0 s in VGX rats). A three-way ANOVA on the mean duration of social investigation after treatment (expressed as percentage of baseline) revealed a significant effect of the dose (F(3, 39)57.9, P,0.001) and treatment factors (F(1, 39)590.3, P,0.001), and a significant effect of the doses3treatment (F(3, 39)512.3, P,0.001) and surgery3treatment (F(1, 39)56.4, P,0.05) interactions. Post-hoc comparisons of individual group means showed that IL-1-induced decreases in social investigation were more marked in sham (58.4% decrease) than in VGX animals (37.2% decrease) (mean duration of social investigation in vehicle-treated sham: 104.865 s vs. IL-1 treated sham: 43.668 s; vehicle treated VGX: 92.463 s vs.
Fig. 2. Effects of vagotomy on IL-1b induced decrease of social exploration: IL-1 (1–30 mg / kg) or saline were injected after the baseline session and rats were tested 2 and 4 later. Each point represents the integrated data from the two time points tested and is presented as mean duration (6S.E.M) of social exploration expressed as percentage of baseline, [n56 / group except for the group injected with IL-1 (3 mg / kg), n55]. The insert shows the mean duration of social investigation (% of baseline) over 4 h for each experimental group (sham vs. VGX) and for each treatment (Vehicle vs. IL-1b).
IL-1 treated VGX: 58.069 s) (F1, 90)54.1, P,0.05) see Fig. 2.
4. Discussion Previous studies on the role of the vagus nerve in neuroimmune interaction have indicated that the vagus is an important pathway of communication between the periphery and the brain during infection or inflammation (Dantzer, 1994; Maier et al., 1998). Our data support this hypothesis and demonstrate that subdiaphragmatic section of the vagus nerve abrogates the depressive effects of IL-1b on social exploration. In contrast, subdiaphragmatic vagotomy failed to alter the febrile responses to the same stimulus (IL-1b), indicating that different mechanisms are involved in fever and altered social exploration behaviour. Our results from the fever study are difficult to reconcile in the face of the mounting evidence indicating the direct involvement of the vagus nerve in propagating the febrile response after infection (Sehic and Blatteis, 1996; Watkins et al., 1995b). Our earlier preliminary observations have indicated that LPS or IL-b induced fever are not attenuated in VGX rats, after intraperitoneal administration of the stimulus. In those studies a single (supramaximal) dose was used which may have been responsible for our failure to observe a reduced febrile response after vagotomy, as the dose of the stimulus was demonstrated to be a factor (Gaykema et al., 1995; Hansen and Krueger, 1997; Kapas et al., 1998). In these studies the influence of the vagus was overridden by increasing the dose of the stimulus, in this case LPS or IL-1b. In the present study, experiments were designed specifically to address this point, so that a dose range of IL-1b was used both in the fever study (0.1–1.0 mg / kg, i.p.) and behaviour (1.0–30.0 mg / kg, i.p.). The results from the fever study demonstrate unequivocally that the magnitude of the response is not affected in the VGX rats and that increasing the dose of IL-1b from 0.1 (sub-threshold) to 1.0 mg / kg (super-maximal) was not a factor. Apart from using a range of doses, the only difference between our study and those of others is that a species specific cytokine was used (rat recombinant IL-b) as compared with human (Watkins et al., 1995b; Fleshner et al., 1998; Opp and Toth, 1998). This however should not be a significant factor as it is unlikely that species specific cytokines should induce responses of different characteristics in different species. In fact direct comparison of the reagents used in different studies may be inappropriate, as ultimately this depends on the biological activity of each preparation as assessed by international units (IU) of activity. Although the magnitude of the febrile responses between the VGX and sham control animals was similar, there were two important differences. Firstly, the basal temperature of the VGX animals was consistently and significantly (P,0.01) different. This has been reported
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previously (Opp and Toth, 1998), and although interesting, a lower basal temperature in VGX animals has not affected the ability of those animals to generate a febrile response of the same magnitude as their sham controls. The second difference was the observation that VGX animals failed to maintain the fever, which began subsiding approximately 2.5 h after the stimulus. In the case of the highest dose (1.0 mg / kg) this difference may be indicative of an involvement of the vagus nerve in maintaining an already established febrile response. The question then arises, if the vagus nerve is not involved in initiating the febrile response, what is the alternative mechanism? One possibility is a humoral, circulating factor and there is a plethora of evidence implicating a blood borne mechanism of IL-1 action in fever. For example, IL-1 induces the release of IL-6, which can interact with the brain via areas devoid of the blood brain barrier (Blatteis, 1992) or through an active transport mechanism (Banks et al., 1994). Unlike IL-1, concentrations of bioactive IL-6 increase dramatically both in the circulation and cerebral spinal fluid of febrile experimental animals (Luheshi et al., 1996). Recent studies in genetically modified animals support the role of IL-6 as a circulating pyrogen (Chai et al., 1996). This study reported that mice with a null mutation in the IL-6 gene failed to exhibit a febrile response to either systemic or icv injection of LPS or recombinant murine IL-1b while responding to an i.c.v. injection of IL-6. These data suggest that IL-6 is an important component of the fever response to exogenous immune stimuli. Our own recent studies have confirmed this by demonstrating total inhibition of systemic LPS induced fever in rats pre-injected with anti-IL-6 antiserum (i.p.) (Cartmell et al., 2000). Other evidence for the involvement of a humoral factor in the mediation of fever was also reported by Takahashi et al. (Takahashi et al., 1997). These workers demonstrated that CIRCUMVENTRICULAL organs such as the subfornical which lie outside the BBB, respond to circulating pyrogen and through their efferent projections activate central pathways involved in fever. Others, suggested that an alternative mechanism to the vagal transmission could be involved in mediating the fever responses in animals treated with high but not low doses of LPS or IL-1b (Romanovsky et al., 1997), or following burn injury in rats (Caldwell et al., 1999b). It is likely therefore that circulating pyrogens, coupled with the classical hypothesis regarding the involvement of circumventricular organs or induction of brain cytokines via activation of receptors on the BBB, still represent the most plausible explanation of interaction between the immune and the central nervous systems in fever. The results presented here indicate that unlike fever, the attenuation of social exploration behaviour induced by peripheral injection of IL-1b is dependent, at least in part, on direct activation of brain cytokines via triggering of the vagus nerve. In the study of Laye et al. (Laye et al., 1995), IL-1b mRNA was shown to be expressed in a number of
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discrete regions in the mouse brain following intraperitoneal injection of LPS and this expression correlated well with changes in behaviour. It was further demonstrated that the induction of this message was inhibited following vagotomy. The results from the present study add support to these findings and indicate that in behaviour, unlike fever, the vagal afferent is a source of peripheral signalling to the brain following an immune challenge. In the brain, the two responses are likely to involve different mechanisms. In the case of fever, IL-1 acts via the release of IL-6 (Klir et al., 1994; Rothwell et al., 1991), whereas depressed social exploration behaviour is independent of IL-6 action (Lenczowski et al., 1999). Other differences include the involvement of corticotrophin releasing hormone (CRH), which mediates brain IL-1 action in fever (Rothwell, 1989) but not behaviour (Bluthe et al., 1989). These differences might originate from the periphery, in the form of different peripheral signals, which then trigger different pathways to the brain to elicit fever or changes in behaviour. Apart from differences in brain mechanisms it is also plausible to suggest that failure to observe reductions in body temperature in the VGX animals in our study could be due to reduction in heat loss known to occur in behaviourally depressed animals after infection (Hart, 1988). This will in turn contribute to the maintenance of an elevated body temperature and consequently the fever observed in these experiments. Although possible, this hypothesis does not address the fact that failure to attenuate the febrile responses in VGX animals in our study was observed at doses not sufficient to induce depressed social exploration behaviour. This would suggest that differences in brain mechanisms could be significant depending on the dose of the inflammatory stimulus used systemically.
5. Conclusions The results from this study demonstrate that a functional dissociation exists between the mechanisms involved in triggering fever and depressed social exploration behaviour after a peripheral immune stimulus. Although the precise nature of this difference remains unknown, our data indicate that whereas induction of social exploration behaviour after a peripheral stimulus is dependent on the functional integrity of the vagus nerve, fever involves alternative mechanisms such as circulating pyrogens.
Acknowledgements This work was supported by the European Community BIOMED-1 concerted action programme ‘Cytokines in the brain’. Thanks are due to, Arnaud Aubert for experimental assistance and Carmen Motard for technical assistance.
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Vagotomy attenuates the behavioural but not the pyrogenic effects of interleukin-1 in rats Giamal N. Luheshi a , *, Rose-Marie Bluthe´ b , David Rushforth a , Nicholas Mulcahy a , Jan-Pieter Konsman b , Michael Goldbach a , Robert Dantzer b a
School of Biological Sciences, University of Manchester, Manchester M13 9 PT, UK b ¨ , 33077 Bordeaux cedex, France INRA-INSERM U394, rue Camille Saint-Saens Received 4 February 2000; accepted 23 May 2000
Abstract Vagal afferent signals, have been implicated in cytokine mediated interactions between the periphery and the central nervous system. Studies in experimental animals have shown that cytokine induced activation of brain mediated responses to infection such as fever, sickness behaviour and pituitary-adrenal activation, are inhibited by subdiaphragmatic vagotomy. We have previously proposed that the peripheral signal to the brain in fever is of a humoral nature while others have suggested that either neural afferents or a mixture of both humoral and neural signals may be involved. The objective of the present study was to examine further the role of vagal transmission, in mediating the febrile response to a systemic injection of IL-1b in rats and to compare this with changes in social exploration behaviour. Intraperitoneal injection of IL-1b (1.0–30.0 mg / kg) inhibited social exploration in rats and this was attenuated in vagotomized animals. Injection of increasing concentrations of IL-1b (0.1–1.0 mg / rat) induced significant (P,0.001) increases in core body temperature. However, in contrast to effects on social exploration, the increase in temperature was not inhibited by vagotomy at any of the doses used. These observations demonstrate a dissociation between the two brain mediated events, one of which is dependent on the integrity of the vagus nerve (social exploration) while the other (fever) is apparently generated by different mechanisms which may include circulating pyrogens. 2000 Elsevier Science B.V. All rights reserved. Keywords: Fever; Cytokines; CNS; Social interaction
1. Introduction Cytokines such as interleukin (IL)-1b are key mediators of the interaction between the immune and central nervous system (CNS) (Hopkins and Rothwell, 1995). Systemic administration of IL-1b in experimental animals results in the same CNS mediated responses associated with sickness behaviour (Rothwell, 1991) which include fever and decrease in social interaction behaviour. The mechanism by which systemically produced large hydrophilic molecules such as IL-1b interact with the brain is controversial but a number of hypothesis have been proposed. These include an active transport mechanism (Banks et al., 1991), access through areas devoid of the blood brain barrier (BBB) such as organum vasculosum of the laminae *Corresponding author. Present address. Douglas Hospital Research Centre, McGill University, 6875 Boulevard LaSalle, Verdun (Quebec), Canada H4H 1R3. Tel.: 11-514-761-6131; fax: 11-514-762-3034. E-mail address: [email protected] (G.N. Luheshi).
terminalis (OVLT) (Blatteis, 1992), or action on receptors situated on endothelial cells lining cerebral blood vessels, resulting in the release of secondary mediators (Van Dam et al., 1996). More recently, the involvement of an afferent neuronal link from the periphery to the brain has gained prominence. A number of recent reports have demonstrated a role for the vagus nerve in transmitting cytokine signals to the brain to elicit an array of host defence responses. These responses, which included fever, were shown to be significantly inhibited in vagotomized (VGX) experimental animals injected peripherally with inflammatory stimuli including IL-1 [see (Watkins et al., 1995a; Maier et al., 1998) for extensive reviews of this area]. However other studies on fever have demonstrated the importance of mechanisms other than neuronal afferents. The most likely of these is the involvement of circulating pyrogens such as IL-6, which has been shown to increase dramatically in the circulation of febrile animals after treatment with, for example LPS (Luheshi et al., 1996; Miller et al., 1997).
1566-0702 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S1566-0702( 00 )00231-9
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It has been suggested that the attenuation of fever and other responses observed in VGX animals may be dependent on the dose of the inflammatory stimulus used (Gaykema et al., 1995; Hansen and Krueger, 1997). Hansen and Kruger (Hansen and Krueger, 1997) demonstrated that increasing the dose of i.p. IL-1b overrides the attenuation in brain temperature and non-rapid eye movement sleep (NREMS) observed in VGX animals. These observations suggested that a pathway other than the vagal afferent is involved in mediating the IL-1b signal from the periphery to the brain. Other workers (Romanovsky et al., 1997) have shown that in VGX animals injected with increasing concentrations of i.v. LPS only the monophasic fever resulting from a low dose of LPS was attenuated while the biphasic fever (in response to higher doses of LPS) remained unaffected by vagotomy. These workers concluded that vagal afferents might be important in mediating responses to minor concentrations of LPS, whereas the response to a higher dose may be mediated by non-vagal mechanisms. Other studies demonstrated that selective vagal rootlet deafferentation failed to completely block the suppression of food intake induced by either LPS or IL-1b administered systemically (Schwartz et al., 1997). Our own preliminary studies (Luheshi, 1998) indicated that fever was not attenuated in VGX animals following i.p. administration of a single dose of either LPS or IL-1b. Other workers (Caldwell et al., 1999a) arrived to a similar conclusion following burn injury in rats. It is obvious from the number of studies performed in this area that conflicting data exists. The majority of studies quite clearly suggest that the vagus plays an important part in mediating cytokine activated signals to the brain, however depending on the dose of the stimulus used or the parameter being measured, this part may not be exclusive and other mediators may be involved. The aim of the present investigation therefore, was to address some of these discrepancies, by (1) performing dose response studies using IL-b administered systemically (i.p.) to study the role of the vagus nerve in the development of fever; and (2) by using the same approach, compare the fever responses with those to changes in social behaviour, a parameter that has been consistently shown to be attenuated by vagal DEAFFRENTIATION.
2. Methods
2.1. Animals All experiments were performed on adult, male, Wistar rats (Charles River, France and Charles River, UK) of 225–250 g body weight. The animals were housed in a temperature controlled room (21.062.08C) artificially lit from 08:00 A.M. to 08:00 P.M., and were provided with food (Extralabo, Provins, France or Beekay international, UK) and water ad libitum. Juvenile male rats (21–35 days
of age) of the same strain served as stimulus animals for the behaviour studies.
2.2. Surgery Animals were food deprived for 24 h prior to surgery and randomly assigned to undergo either vagotomy or sham operation as previously described by Goldbach et al. (Goldbach et al., 1997) modified for the rat. Briefly, animals were anaesthetised with a mixture of ketamine and xylazine (61.0 and 9.0 mg kg 21 respectively), and the main ventral and dorsal trunks of the vagus nerve were transected immediately above the stomach. All connective tissue 2–3 cm between the gastric artery and the oesophagus was removed to ensure the transection of any accessory vagal branches. Sham animals underwent the same surgical procedure excluding transection of the nerve. All rats received a single intramuscular injection of AMOXCILLIN (75.0 mg kg 21 ) and were allowed to recover from surgery for 4 weeks before experimentation. The surgery was performed by the same investigator for both the fever and behaviour studies. Vagotomy was verified by isolectin I B4 (1 mg / ml phosphate buffered saline; L5391, Sigma) binding in the NTS. Most vagal afferents project to the NTS and isolectin I B4 has the peculiarity of binding to vagal endings in this region (Li et al., 1997). The distribution of isolectin I B4 in the NTS of sham operated rats was similar to that reported previously. High isolectin I B4 binding was found in the medial and commissural nuclei as well as in the NTS of shamoperated animals. After subdiaphragmatic vagotomy, isolectin I B4 binding in these structures was highly reduced (data not shown).
2.3. Experimental Hand-held, lightly-restrained rats, were injected i.p., (1.0 ml / kg) with rat recombinant IL-1b (biological activity: 317 IU / mg, Dr S. Poole, NIBSC, Potters Bar, UK) dissolved in pyrogen free saline containing 0.1% endotoxin-free bovine serum albumin (BSA, A-8806, Sigma, USA) and using a dose range of 0.1–1.0 mg / kg for fever studies and 1.0–30.0 mg / kg for behaviour (Anforth et al., 1998). Control animals received an equal volume of 0.9% sterile saline. Behaviour studies were conducted as a between–within comparison, the surgery (sham versus vagotomy) and dose (1.0, 3.0, 10.0 and 30.0 mg / kg) factors were considered as between factors; the treatment (IL-1b versus vehicle) was considered as a within factor.
2.3.1. Fever Core body temperature was measured continuously in conscious, undisturbed, individually housed animals (n5 10–15) by remote radio-telemetry, via battery-operated biotelemetry transmitters (Data Sciences, St. Paul, USA), previously implanted in the abdominal cavity during the
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vagotomy or sham procedures. The output frequency (Hz) of each transmitter was monitored by an antenna mounted in a receiver board situated beneath the cage of each animal, and channelled to a peripheral processor (Dataquest IV, Data Sciences). Frequencies were sampled at 10-min intervals and converted to degrees Celsius (8C). Animals received a single injection of IL-1b or vehicle at 10:00 A.M. and their temperatures were monitored for 7 h.
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sponded to the lowest dose of IL-1b (0.1 mg, Fig. 1a) while increasing the dose to 0.5 (Fig. 1b) and 1.0 mg / kg (Fig. 1c) resulted in a highly significant febrile response in both groups (P,0.001) of animals when compared to their vehicle treated controls. In both cases, a stress induced transient rise in temperature was followed by a sharp increase, which peaked at 2.5 h after which it declined
2.3.2. Social exploration IL-1b-induced sickness behaviour was assessed in sham (n524) and VGX (n524) rats by the reduction in duration of social investigation of a conspecific juvenile introduced momentarily into the home cage of the test animal at various times before and after i.p. injection of either IL-1b or vehicle. Social investigation was measured by the duration of ano-genital sniffing and close following of the juvenile during a 4-min session immediately before the injection and 2 and 4 h later. Different juveniles were presented on each occasion. 2.3.3. Statistical analysis Data from fever experiments are presented as means6S.E.M. Temperature data were analysed as area under the curve (AUC) and one-way ANOVA followed by Scheffe’s post-hoc test. In the behaviour studies in order to verify that surgery did not affect the behaviour of VGX animals compared with sham-operated animals, duration of social investigation (seconds) measured during the first session before injection of vehicle or IL-1b was submitted to a one-way ANOVA. The mean duration of social investigation over the 4 h period following injection of vehicle or IL-1b was expressed as percentage of baseline and submitted to a three-way ANOVA, doses of IL-1b and surgery as between factors and treatment as within factor. Post-hoc comparisons of individual group means were carried out by the protected least, significant difference test (LSD).
3. Results
3.1. Effect of subdiaphragmatic vagotomy on IL-1b induced fever The basal temperatures of animals used in these experiments ranged from 36.58C to 36.88C and were consistently lower in the VGX (36.560.18C) compared with the sham controls (36.860.18C, P,0.01, n536). The basal temperatures rose moderately in all rats at the beginning of each experiment, probably due to handling stress during injection. This initial rise in temperature normally persisted for 1 h, after which it returned to basal levels. Injection of IL-1b (0.1–1.0 mg / kg, i.p.) induced a dose dependent monophasic fever in both sham and VGX animals. Neither VGX, nor sham operated animals re-
Fig. 1. Effect of vagotomy on core body temperature: Changes in core body temperature of vagotomized and sham operated rats after i.p. injection of different doses of IL-1 (n510–15) or vehicle (1.0 ml / kg, n510–15) at time 0 h. Values are means6S.E.M (n510–15) and were analysed using area under the curve (AUC;8C / min).
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towards basal levels (Fig. 1b and c). There was no difference in the magnitude of the febrile response to IL-1b, exhibited by either VGX or the sham animals. However, VGX animals injected with 1.0 mg / kg IL-1b failed to sustain febrile response compared to counterpart sham controls (Fig. 1c). In this case the fever peaked at 2.5 h after which it declined more rapidly towards basal temperature when compared with the shams where the rise in temperature was maintained for up to 5 h.
3.2. Effect of subdiaphragmatic vagotomy on IL-1b induced depression in social exploration A one-way ANOVA on the duration of social investigation measured immediately prior to treatment did not reveal any difference between VGX and sham rats (mean duration of investigation: 127.965.0 s in sham vs. 137.264.0 s in VGX rats). A three-way ANOVA on the mean duration of social investigation after treatment (expressed as percentage of baseline) revealed a significant effect of the dose (F(3, 39)57.9, P,0.001) and treatment factors (F(1, 39)590.3, P,0.001), and a significant effect of the doses3treatment (F(3, 39)512.3, P,0.001) and surgery3treatment (F(1, 39)56.4, P,0.05) interactions. Post-hoc comparisons of individual group means showed that IL-1-induced decreases in social investigation were more marked in sham (58.4% decrease) than in VGX animals (37.2% decrease) (mean duration of social investigation in vehicle-treated sham: 104.865 s vs. IL-1 treated sham: 43.668 s; vehicle treated VGX: 92.463 s vs.
Fig. 2. Effects of vagotomy on IL-1b induced decrease of social exploration: IL-1 (1–30 mg / kg) or saline were injected after the baseline session and rats were tested 2 and 4 later. Each point represents the integrated data from the two time points tested and is presented as mean duration (6S.E.M) of social exploration expressed as percentage of baseline, [n56 / group except for the group injected with IL-1 (3 mg / kg), n55]. The insert shows the mean duration of social investigation (% of baseline) over 4 h for each experimental group (sham vs. VGX) and for each treatment (Vehicle vs. IL-1b).
IL-1 treated VGX: 58.069 s) (F1, 90)54.1, P,0.05) see Fig. 2.
4. Discussion Previous studies on the role of the vagus nerve in neuroimmune interaction have indicated that the vagus is an important pathway of communication between the periphery and the brain during infection or inflammation (Dantzer, 1994; Maier et al., 1998). Our data support this hypothesis and demonstrate that subdiaphragmatic section of the vagus nerve abrogates the depressive effects of IL-1b on social exploration. In contrast, subdiaphragmatic vagotomy failed to alter the febrile responses to the same stimulus (IL-1b), indicating that different mechanisms are involved in fever and altered social exploration behaviour. Our results from the fever study are difficult to reconcile in the face of the mounting evidence indicating the direct involvement of the vagus nerve in propagating the febrile response after infection (Sehic and Blatteis, 1996; Watkins et al., 1995b). Our earlier preliminary observations have indicated that LPS or IL-b induced fever are not attenuated in VGX rats, after intraperitoneal administration of the stimulus. In those studies a single (supramaximal) dose was used which may have been responsible for our failure to observe a reduced febrile response after vagotomy, as the dose of the stimulus was demonstrated to be a factor (Gaykema et al., 1995; Hansen and Krueger, 1997; Kapas et al., 1998). In these studies the influence of the vagus was overridden by increasing the dose of the stimulus, in this case LPS or IL-1b. In the present study, experiments were designed specifically to address this point, so that a dose range of IL-1b was used both in the fever study (0.1–1.0 mg / kg, i.p.) and behaviour (1.0–30.0 mg / kg, i.p.). The results from the fever study demonstrate unequivocally that the magnitude of the response is not affected in the VGX rats and that increasing the dose of IL-1b from 0.1 (sub-threshold) to 1.0 mg / kg (super-maximal) was not a factor. Apart from using a range of doses, the only difference between our study and those of others is that a species specific cytokine was used (rat recombinant IL-b) as compared with human (Watkins et al., 1995b; Fleshner et al., 1998; Opp and Toth, 1998). This however should not be a significant factor as it is unlikely that species specific cytokines should induce responses of different characteristics in different species. In fact direct comparison of the reagents used in different studies may be inappropriate, as ultimately this depends on the biological activity of each preparation as assessed by international units (IU) of activity. Although the magnitude of the febrile responses between the VGX and sham control animals was similar, there were two important differences. Firstly, the basal temperature of the VGX animals was consistently and significantly (P,0.01) different. This has been reported
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previously (Opp and Toth, 1998), and although interesting, a lower basal temperature in VGX animals has not affected the ability of those animals to generate a febrile response of the same magnitude as their sham controls. The second difference was the observation that VGX animals failed to maintain the fever, which began subsiding approximately 2.5 h after the stimulus. In the case of the highest dose (1.0 mg / kg) this difference may be indicative of an involvement of the vagus nerve in maintaining an already established febrile response. The question then arises, if the vagus nerve is not involved in initiating the febrile response, what is the alternative mechanism? One possibility is a humoral, circulating factor and there is a plethora of evidence implicating a blood borne mechanism of IL-1 action in fever. For example, IL-1 induces the release of IL-6, which can interact with the brain via areas devoid of the blood brain barrier (Blatteis, 1992) or through an active transport mechanism (Banks et al., 1994). Unlike IL-1, concentrations of bioactive IL-6 increase dramatically both in the circulation and cerebral spinal fluid of febrile experimental animals (Luheshi et al., 1996). Recent studies in genetically modified animals support the role of IL-6 as a circulating pyrogen (Chai et al., 1996). This study reported that mice with a null mutation in the IL-6 gene failed to exhibit a febrile response to either systemic or icv injection of LPS or recombinant murine IL-1b while responding to an i.c.v. injection of IL-6. These data suggest that IL-6 is an important component of the fever response to exogenous immune stimuli. Our own recent studies have confirmed this by demonstrating total inhibition of systemic LPS induced fever in rats pre-injected with anti-IL-6 antiserum (i.p.) (Cartmell et al., 2000). Other evidence for the involvement of a humoral factor in the mediation of fever was also reported by Takahashi et al. (Takahashi et al., 1997). These workers demonstrated that CIRCUMVENTRICULAL organs such as the subfornical which lie outside the BBB, respond to circulating pyrogen and through their efferent projections activate central pathways involved in fever. Others, suggested that an alternative mechanism to the vagal transmission could be involved in mediating the fever responses in animals treated with high but not low doses of LPS or IL-1b (Romanovsky et al., 1997), or following burn injury in rats (Caldwell et al., 1999b). It is likely therefore that circulating pyrogens, coupled with the classical hypothesis regarding the involvement of circumventricular organs or induction of brain cytokines via activation of receptors on the BBB, still represent the most plausible explanation of interaction between the immune and the central nervous systems in fever. The results presented here indicate that unlike fever, the attenuation of social exploration behaviour induced by peripheral injection of IL-1b is dependent, at least in part, on direct activation of brain cytokines via triggering of the vagus nerve. In the study of Laye et al. (Laye et al., 1995), IL-1b mRNA was shown to be expressed in a number of
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discrete regions in the mouse brain following intraperitoneal injection of LPS and this expression correlated well with changes in behaviour. It was further demonstrated that the induction of this message was inhibited following vagotomy. The results from the present study add support to these findings and indicate that in behaviour, unlike fever, the vagal afferent is a source of peripheral signalling to the brain following an immune challenge. In the brain, the two responses are likely to involve different mechanisms. In the case of fever, IL-1 acts via the release of IL-6 (Klir et al., 1994; Rothwell et al., 1991), whereas depressed social exploration behaviour is independent of IL-6 action (Lenczowski et al., 1999). Other differences include the involvement of corticotrophin releasing hormone (CRH), which mediates brain IL-1 action in fever (Rothwell, 1989) but not behaviour (Bluthe et al., 1989). These differences might originate from the periphery, in the form of different peripheral signals, which then trigger different pathways to the brain to elicit fever or changes in behaviour. Apart from differences in brain mechanisms it is also plausible to suggest that failure to observe reductions in body temperature in the VGX animals in our study could be due to reduction in heat loss known to occur in behaviourally depressed animals after infection (Hart, 1988). This will in turn contribute to the maintenance of an elevated body temperature and consequently the fever observed in these experiments. Although possible, this hypothesis does not address the fact that failure to attenuate the febrile responses in VGX animals in our study was observed at doses not sufficient to induce depressed social exploration behaviour. This would suggest that differences in brain mechanisms could be significant depending on the dose of the inflammatory stimulus used systemically.
5. Conclusions The results from this study demonstrate that a functional dissociation exists between the mechanisms involved in triggering fever and depressed social exploration behaviour after a peripheral immune stimulus. Although the precise nature of this difference remains unknown, our data indicate that whereas induction of social exploration behaviour after a peripheral stimulus is dependent on the functional integrity of the vagus nerve, fever involves alternative mechanisms such as circulating pyrogens.
Acknowledgements This work was supported by the European Community BIOMED-1 concerted action programme ‘Cytokines in the brain’. Thanks are due to, Arnaud Aubert for experimental assistance and Carmen Motard for technical assistance.
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