Pain 78 (1998) 181–190
Lipopolysaccharide-induced hyperalgesia of intracranial capsaicin sensitive afferents in conscious rats Richard H.A. Kemper a , b ,*, Mary B. Spoelstra a , b, Willem J. Meijler a, Gert J. Ter Horst b a
Department of Psychiatry and Anesthesiology, University Hospital Groningen, Hanzeplein 1, 9713 EZ Groningen, The Netherlands b Pain Centre, University Hospital Groningen, Hanzeplein 1, 9713 EZ Groningen, The Netherlands Received 9 March 1998; received in revised form 3 June 1998; accepted 9 July 1998
Abstract Migraineous and non-migraineous headache is reported to be at highest intensity after an infection. This study investigated whether activation of the immune system can induce hyperalgesia in intracranial capsaicin sensitive afferents. The effects of intraperitoneal injected lipopolysaccharides (LPS) on behavior and c-fos expression in the trigeminal nucleus caudalis layer I, II (TNC I,II) elicited by intracisternally applied capsaicin were studied. Low concentrations of LPS potentiated capsaicin-induced immobilization behavior without affecting c-fos expression in the TNC I,II. Large amounts of LPS however increased the number of capsaicin-induced c-fos positive cells in the TNC I,II. These effects of LPS on capsaicin sensitive afferents are probably mediated by cytokines that act at peripheral vagal nerves, central brain regions or via direct actions of cytokines on capsaicin sensitive afferent nerve terminals. The hyperalgesic action of LPS on intracranial trigeminal and possibly other capsaicin sensitive afferents of the head may explain why different types of infections are accompanied by headache and why migraineous and non-migraineous headache is of highest intensity after an infection. 1998 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Headache; Migraine; Hyperalgesia; Capsaicin; Lipopolysaccharides; Inflammation
1. Introduction Stress, fatigue and menstrual periods are well known precipitators of migraine. Less well known is that infections also can precipitate migraine and that infections give headache of highest pain intensity in both migraineurs and nonmigraineurs when compared to pain induced by other precipitators (Chabriat et al., 1997). There is also evidence that the immune system of migraine patients is different compared to non-migraineurs (Covelli et al., 1990a; Covelli et al., 1990b; Covelli et al., 1991a; Covelli et al., 1991b). It is not clear how infections trigger migraine or how they can enhance pain intensity. A possible explanation for increased pain intensity is that intracranial trigeminal sensory nerves become hyperalgesic after an immunological challenge. * Corresponding author. Department of Biological Psychiatry, rm. 7,25, Academic Hospital Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands. Tel.: +31 503 612094; fax: +31 503 611699; e-mail:
[email protected]
0304-3959/98/$ - see front matter PII S0304-3959 (98 )0 0125-0
This induction of hyperalgesia by components of the immune system has been shown for several peripheral sensory nerves. Cytokines like tumor necrosis factor alpha (TNF-a), interleukin 6 and interleukin 1-b(IL-1b) are able to induce hyperalgesia in a nociception model that uses mechanical stimulation of the hind-paw of the rat (Ferreira et al., 1988; Cunha et al., 1992). Injection of these cytokines reduced the reaction time for rats to respond to mechanical pressure applied to the hind paw. Moreover capsaicininduced vasodilatation in the rat skin, which is thought to be mediated by nociceptive afferents, could be enhanced by IL-1b (Herbert and Holzer, 1994). Furthermore, lipopolysaccharides (LPS, LPS are endotoxins of the cell wall of gram-negative bacteria) could facilitate the release of calcitonin gene related peptide (CGRP) from capsaicin sensitive sensory nerves located in the trachea of rats (Hua et al., 1996). Cytokines like IL-1ß and TNF-a are critically involved in this facilitation. None of the above mentioned studies examined effects of inflammation on intracranial trigeminal sensory nerves. Tri-
1998 International Association for the Study of Pain. Published by Elsevier Science B.V.
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geminal sensory nerves are the primary nociceptive afferents of the head and are generally considered to be involved in headache/migraine pathophysiology (Moskowitz, 1984; Buzzi and Moskowitz, 1992; Goadsby and Edvinsson, 1993). IL-1ß has been shown to increase nociceptive processing in the trigeminal nucleus caudalis (Oka et al., 1993) but these experiments used extracranial trigeminal nerve stimulation in anesthetized rats. The above mentioned observations prompted us to examine whether intracranial sensory nerves are also subject to sensitization after an immunological challenge. This might explain enhanced headache intensity after infection seen both in migraineurs and non-migraineurs (Chabriat et al., 1997). Intracisternal (i.c.) infusion of the irritant capsaicin in conscious rats was used to activate trigeminal nociceptive fibers (Kemper et al., 1997). A low and high dose of intraperitoneally (i.p.) injected LPS was used to stimulate the immune system of the rat. LPS delivered systemically mimics many aspects of bacterial infection including immunological alterations (Morrison, 1987) fever and pain (Watkins et al., 1994b). Capsaicin-induced c-fos protein expression in the outer layers of the trigeminal nucleus caudalis (TNC I, II) was quantified to assess activity of the nociceptive part of the sensory trigeminal system. Behavior shown during and directly after infusion of capsaicin was recorded on videotape and analyzed after the experiment. The c-fos expression in the nucleus of the solitary tract (NTS) and area postrema (AP) was also quantified.
with 0.5% chlorhexidine. All rats were anesthetized with 0.4 ml/kg i.m. hypnorm (fentanyl 0.3 mg/ml and fluanisone 10 mg/ml; Janssen, Beerse, Belgium) and pentobarbital (24 mg/kg i.p.). A midline incision in the skin at the top of the head was made and membranes from the parietal, interparietal and rostrodorsal part of the occipital skull were removed. The Cisterna Magna (CM) cannula was prepared from a stainless steel needle (0.6 × 25 mm, 23 G × 1″; Braun, Melsungen, Germany) which was shortened to 6.5 mm. Rats were placed in a stereotaxic apparatus with incisor bar at −7 mm from the horizontal plane. Two holes were drilled into the caudal corners of the interparietal skull and two screws (diameters 1.0 mm and l.3 mm) were driven 1.5 mm into the skull. A hole (diameter 1.2 mm) was drilled at the midline of the external occipital crest for placement of the CM cannula. The CM cannula was carefully placed through the hole with a horizontal rostro-caudal approach and pushed beneath the dorsal part of the occipital bone until the dorso-caudal part of the occipital bone was reached. Then the cannula was slowly turned from the horizontal, rostral-caudal plane into the dorsal-ventral plane. Guiding the CM cannula along the occipital bone caudal from the cerebellum, it was gently positioned into the Cisterna Magna. Correct placement of the cannula was confirmed by withdrawal of CSF after which the cannula was fixed to the skull with dental cement (Kemdent, Purton Swindon, UK) and closed with a piece of silicon tube. The wound was sutured and rats were allowed to recover for 3 days.
2. Methods
2.3. Experimental procedures A time line drawing for the experiments is shown in Fig.
2.1. Animals Male Wistar rats weighting 308 ± 4.1 g were used. All rats were housed groupwise (3 rats/cage) on a light/dark regime (L/D: 0800 h / 2000 h) and surgery was performed 5 days after arrival. Experiments were approved by, and were under close supervision of, the committee on Animal Bio-Ethics of the University of Groningen (FDC 1191) and performed according to the ethical guidelines for investigations of experimental pain in conscious animals (Zimmermann, 1983). Based on prior experiments (Kemper et al., 1997) in which the 100 nM capsaicin concentration did not significantly augment the c-fos expression in the TNC I,II and 1000 nM gave a maximal activation, we chose to use a 250 nM capsaicin concentration. As rats were sacrificed 2 h after capsaicin treatment, it was ensured that the pain caused by capsaicin was short-lived. None of the animals suffered to such extent (behavioral observations) that they had to be terminated before the end of the experiments. 2.2. Surgical procedures Cannulas, surgical materials and rat skin were disinfected
1. 2.4. Injection Five hours prior to the infusion of capsaicin or vehicle, rats were injected intraperitoneally with either vehicle, 0.75 mg/kg LPS (LPS(L)) or 37.5 mg/kg LPS (LPS(H)). Concentrations of LPS and the period of 5 h were based on the
Fig. 1. Time line drawing of the experiments. A cisterna magna cannula was implanted 3 days prior to the day of experimenting. At day 3 rats were i.p. injected with saline, 0.75 mg/kg LPS (LPS(L)) or 37.5 mg/kg LPS (LPS(H)). After 5 h rats were infused with vehicle or 250 nM capsaicin for 2 min. Behavior was recorded during infusion and the 10 min afterwards. Two hours after infusion rats were perfused.
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study of Hua and colleagues who showed that 0.75 mg/kg LPS enhanced capsaicin-induced CGRP release from sensory nerves of the trachea 5 h after LPS injection (Hua et al., 1996). The higher concentration of 37.5 mg/kg LPS was used to study possible dose dependent effects of LPS. 2.5. Infusion Rats were placed into the experimental cage (30 × 30 × 30 cm) and capsaicin (250 nM) or vehicle was infused into the CM with a microinjection pump (CMA100, Carnegie Medicin, Stockholm, Sweden). Rats received 100 ml capsaicin in 2 min. During the infusion and 10 min thereafter rats were filmed on videotape to allow analysis of behavior afterwards. 2.6. Perfusion and immunocytochemistry Rats were perfused 2 h following infusion of capsaicin or vehicle. Prior to the transcardial perfusion rats were deeply anesthetized with sodium pentobarbital and perfused with 0.9% saline for 1 min, followed by 4% paraformaldehyde (PF) in 0.1 M phosphate-buffered saline (pH 7.4) for 20 min. After removal of the occipital bone, placement of the cannula in the CM was confirmed and extent of the infusion into the epidural space was determined by inspection of the Evans Blue (dissolved 0.2% in the capsaicin and vehicle solutions) staining. After the removal, the brains were post-fixed in 4% PF for 24 h. Brain stem and upper spinal cord were cryoprotected by overnight storage in 30% sucrose in 0.1 M phosphate buffer (pH 7.4). Forty mm thick coronal serial sections were prepared on a cryostat microtome at −15°C, and collected in 0.2 M potassiumphosphate-buffered saline (KPBS, pH 7.4) with sodiumazide (0.1%). Free-floating sections were immunocytochemically stained for c-fos protein according to the following protocol. Sections were rinsed 3 × 10 min in KPBS, pre-treated with 0.3% H2O2 in KPBS for 10 min, rinsed 3 × 10 min in KPBS and pre-incubated in 2% bovine serum albumin (BSA; Merck, Darmstadt, Germany), 2% normal serum (NS, normal rabbit serum Sigma Chemie, Bornem, Belgium) in KPBS for 4 h at room temperature. Subsequently, sections were incubated in 2% BSA, 2% NS and primary antibody sheep-anti-c-fos (1:2000; Cambridge Research Chemicals, Northwich, UK) in KPBS with 0.5% triton X-100 (KPBS-T; Bayer, Deventer, Netherlands) overnight at room temperature. Sections were rinsed 3 × 10 min. in KPBS and incubated in 2% BSA, 2% NS and second antibody (1:200 biotinylated rabbit-a-sheep IgG (Pierce, Rockford)) in KPBS-T at room temperature for 2 h. After 3 × 10 min. washes in KPBS, sections were incubated in avidine-biotine-peroxidase complex (Vector Labs, Burlingame) in KPBS-T with 2% BSA for 2 h at room temperature. Hereafter, sections were washed in 3 × 10 min. KPBS and 2 × 10 min. in 0.1 M sodiumacetate buffer (NaAc, pH
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6.0). For the final staining procedure 3.3′-diaminobenzidine tetrahydrochloride (0.05%) and ammoniumchloride (0.04%) were dissolved in 1/2 v distilled water and 1/2 v NAS solution (5% NikkelAmmoniumSulfate dissolved in 4/ 5 v 0.2M NaAc and 1/5 v distilled water). To start the diaminobenzidine reaction 0.3% H2O2 was added. The reaction was stopped after 20 min. Sections were washed 2 × 10 min. in 0.1M NaAc and 3 × 10 min. in KPBS, mounted on gelatin coated slides, air dried, dehydrated in graded ethanols and xylol and cover-slipped with DEPEX. All staining procedures were with gentle agitation. 2.7. Quantification 2.7.1. TNC layer I, II C-fos immunoreactive cells were counted at −1, −2, −3, −4, −5 and −6 mm caudal from obex by an observer blinded from experimental procedures. Sections from −0.5 to −1.5 mm were averaged to obtain the count for the −1 mm level and so on. To obtain accurate sampling of sections for each level, the trigeminal nucleus of one rat was dissected (40 mm, freezing microtome) from obex to −7 mm from obex and all sections were immediately mounted on gelatin coated slides. A Nissl staining was performed to show cytoarchitecture of the sections and the c-fos stained sections were compared to these sections to determine exact distance of the c-fos expression from obex. As there were no significant differences in the number of c-fos positive cells between the right or left side of the TNC I,II, the total number of cells per section was counted. The mean of the total TNC I,II was calculated by averaging the c-fos expression at the six levels. 2.7.2. NTS and AP As of the functional differences between both the medial/ lateral and the rostral/caudal parts of the nucleus of the solitary tract (NTS), c-fos positive cells were counted in the lateral and medial divisions of the NTS at the level of the obex (bregma −13.68 mm) and also more rostrally at bregma −11.6 mm (Paxinos and Watson, 1997). The Area Postrema (AP) was counted at the level of the obex (bregma −13.68 mm (Paxinos and Watson, 1997)). Three sections of each part of the NTS and AP were counted and averaged for each animal. 2.8. Weight-loss and temperature To assess whether the different concentrations of LPS had an effect on body-weight or temperature, rats were weighed before the injection of vehicle or LPS and 5 h later just before the infusion of capsaicin or vehicle. Rectal temperature was measured 7 h after the i.p. injection of 37.5 mg/kg LPS, 0.75 mg/kg LPS or vehicle during the deep anesthetisation necessary for perfusion. Temperature was not measured at earlier time-points to prevent interference with the measurement of c-fos expression and behavior.
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2.9. Behavior Videotapes of behavior shown during the 2 min of infusion and the 10 min post-infusion were analyzed with dedicated software (The Observer 3.0, Noldus Information Technology, Wageningen, the Netherlands). As rats had to be moved from the experimental to the home-cage after the infusion, the behavior exhibited during the 2 min directly after the infusion were not analyzed. The remaining 8 min were analyzed in two periods of 4 min (3–7 and 7–11 min after infusion) to observe if possible initial behavioral differences remained present. Three major types of behavior elements were distinguished in the analysis; exploring, immobilization and discomfort behavior (Table 1). Occasionally, the animals also showed resting, burying, feeding and scratching/grooming of the body.
that received i.c. vehicle and amongst the three groups of animals that received i.c. capsaicin. The statistical software package Sigmastat (Jandel Scientific, San Rafael) was used to analyze the data. The One Way ANOVA with Student–Neuman Keuls test as multiple comparison method (pairwise) was used to test the effects of different doses of LPS. Sigmastat tests normal distribution and equal variance within the groups, two requirements for the One Way ANOVA. In cases of non-normal distribution or unequal variance (which occurred occasionally throughout all different behaviors and brain areas that were counted for c-fos positive cells) the non-parametric variant of the One Way ANOVA, the Kruskall–Wallis ANOVA on Ranks with Dunn’s test as multiple comparison (pairwise) was performed. P , 0.05 was considered significant.
2.10. D . rugs
3. Results
The capsaicin stock solution (3.05 mg capsaicin per 1 ml of vehicle (saline-ethanol-Tween80 (8:1:1)) was diluted 1:40 in saline to which 0.2% Evans Blue (Merck, Darmstadt) was added. This yields the 250 nM capsaicin concentration. Previous experiments (Kemper et al., 1997) showed that i.c. infused 100 nM capsaicin concentration could not activate the TNC I,II whereas 1000 nM i.c. capsaicin gave a maximal activation. To be able to observe LPS modulation of capsaicin-induced c-fos expression in the TNC I,II the intermediate 250 nM concentration was used in this experiment. LPS (E. Coli Serotype 0.26:B6; Sigma Chemie, Bornem, Belgium) was dissolved in saline and injected intraperitoneally (i.p.) in concentrations of 0.75 mg/kg LPS (LPS(L)) or 37.5 mg/kg LPS (LPS(H))
3.1. LPS; appearance, weight-loss and temperature
2.11. .Statistical analyses To elucidate the effects of LPS, statistical analysis was performed separately amongst the three groups of animals Table 1 Behaviors that were analyzed during intracisternal infusion of vehicle or capsaicin Exploring
Immobilization Discomfort
Head grooming Head scratching Escape behavior
Sniffing and slowly moving around the cage to explore the (new) environment. Occasionally standing on the hind-paws (rearing) All forms of complete immobilization excluding resting Three active behaviors that were interpreted by the investigator as signs of discomfort that were introduced by the capsaicin infusion into the CM (Kemper et al., 1997) were included in this behavior Licking of the fore-paws and washing the head Licking of the fore- or hind-paws and scratching of the head Rapid moving, turning and rearing, maybe jumping, trying to get out of the cage
Prior to i.c. infusion with capsaicin or vehicle, animals injected with LPS(H) showed signs of illness including piloerection and inactivity. LPS(L) treated animals did not show any signs of illness prior to infusion. LPS(L) and LPS(H) injected animals both showed significantly more weight-loss compared to saline injected animals (7.0 ± 0.5 g, 6.5 ± 0.6 g and 4.1 ± 0.6 g, respectively, see Fig. 2). No significant differences in body temperature could be observed between animals injected with 37.5 mg/ kg LPS, 0.75 mg/kg LPS and saline (37.8 ± 0.2°C, 37.8 ± 0.2°C and 37.5 ± 0.1°C, respectively). 3.2. Behavior The five behaviors measured (immobilization, exploring, head scratching head grooming and escape behavior) accounted for 90.5% of all the behaviors shown during and after i.c. infusion with vehicle or capsaicin. During capsaicin infusion, escape behavior was the main discomfort behavior whereas after infusion, grooming and scratching of the head were the most shown discomfort behaviors. Primary remaining behaviors were resting and body grooming which were especially shown by the LPS(L) + Vehicle and the Control group, respectively. Occasionally the animals also showed eating, drinking, burying and scratching of the body. 3.3. Exploring behavior As is shown in Fig. 3, i.c. capsaicin significantly decreases the time animals spend on exploring behavior in all three periods compared to i.c. vehicle infused animals. The time spend on exploring behavior in animals treated with LPS(L) + Vehicle is significantly reduced 3–7 min after infusion (85.4 ± 15.6 s vs. Control: 181.6 ± 29.6 s)
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Fig. 2. Weight-loss (mean ± SEM) from i.p. injection of either saline, 0.75 mg/kg LPS (LPS(L)) or 37.5 mg/kg LPS (LPS(H)) until infusion of vehicle or capsaicin 5 h later. *significantly different from saline (P , 0.05).
whereas it is reduced during and directly after infusion of vehicle in the LPS(H) + Vehicle group (52.8 ± 12.0 s and 72.4 ± 23.8 s vs. Control: 109.6 ± 5.5 s and 181.6 ± 29.6 s, respectively). The LPS(H)-induced changes in the time spend on exploring behavior have disappeared 7–11 min after infusion. Thus differences in exploring behavior caused by LPS(H) are transient. The LPS(H) + Caps treated animals display a significant decrease of the time spend on exploring behavior both during and 3–7 min after the i.c. capsaicin infusion when compared to the Saline + Caps group. There is no difference in the time spend on exploring behavior between the LPS(L) + Caps and the Saline + Caps groups in any of the measured periods. 3.4. Immobilization Immobilization behavior was induced especially in the LPS(H) treated animals. Animals that were treated with the high concentrations of LPS were immobilizing more than 60% of the time during i.c. infusion of either vehicle or capsaicin. In the post-infusion periods this percentage even increased. There were no significant differences in the time spend on immobilization behavior between control rats and rats treated with LPS(L) + Vehicle in any of the measured periods. However, during and 3–7 min after the i.c. infusion of capsaicin the LPS(L) + Caps group showed increase in the period spent on immobilization behavior (46.6 ± 8.1 s and 184.8 ± 31.6 s, respectively) compared to Saline + Caps treated animals (24.2 ± 7.2 s and 89.9 ± 24.2 s, respectively). LPS(L) thus potentiated the time spend on capsaicin-induced immobilization behavior in these two periods.
Fig. 3. Time spend on different kind of behaviors (mean ± SEM) observed during the 2 min of infusion of either capsaicin 250 nM (Caps) or vehicle (control) and in two subsequent periods of 4 min thereafter. Groups LPS(L) and LPS(H) received an i.p. injection with 0.75 mg/kg LPS or 37.5 mg/kg LPS, respectively 5 h prior to infusion. N.S. means value is 0 and therefore not shown. (A) Exploring behavior. (B) Immobilization behavior. (C) Discomfort behavior. 1, 2, 3 and 4 are significantly different from control, LPS(L) + Vehicle, Saline + Caps and LPS(L) + Caps, respectively, (P , 0.05).
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(103 ± 22) compared to the control group. The LPS (L) + Caps group showed no differences in TNC I,II c-fos expression compared to the Saline + Caps group. Moreover, although not significant, it was somewhat decreased in the LPS(L) pretreated animals (61 ± 12). Contrary to the LPS(L) + Caps group, animals treated with LPS(H) + Caps displayed a significant increase in the number of cells expressing c-fos protein (165 ± 20) in layer I, II of the TNC compared to Saline + Caps treated rats. 3.6.2. NTS and AP The number of c-fos positive cells in the AP and the various parts of the NTS is presented in Table 2. The highest (absolute) increase in numbers of cells expressing c-fos caused by both LPS and capsaicin was observed in the caudomedial portion of the NTS adjoining the AP (Control: 4 ± 2, LPS(L) + Vehicle: 51 ± 13, LPS(H) + Vehicle: 152 ± 11, Saline + Caps: 69 ± 38). Combined LPS-capsaicin treatment could not significantly alter the c-fos expression in the NTS and the AP compared to combined saline-capsaicin treated animals. C-fos protein expression in the Area Postrema is significantly increased in the LPS(H) + Vehicle and Saline + Caps treated animals when compared to the control group (33 ± 5, 39 ± 23, 4 ± 3, respectively).
Fig. 4. Number of c-fos protein positive cells (mean ± SEM) in the outer layers of the trigeminal nucleus caudalis (TNC I,II) of animals treated with i.p. vehicle (control), 0.75 mg/kg LPS (LPS0.75) or 37.5 mg/kg LPS (LPS0.75) injection 5 h prior to intracisternally infusion of vehicle (control) or capsaicin 250 nM (C250). 1, 2 and 4 are significantly different from control, LPS0.75 and LPS0.75 + C250, respectively (P , 0.05).
3.5. Discomfort Saline + Caps treated animals significantly spend more time on discomfort behavior (head grooming, head scratching and escape behavior) during (20.7 ± 7.7 s) and 3–7 min after (95.6 ± 30.8 s) i.c. capsaicin infusion compared to Control rats (during infusion: 0 ± 0 s, 3–7 min after infusion: 9.1 ± 5.7 s). No differences in time spend on discomfort behavior are present between LPS(L) + Caps, LPS (H) + Caps and Saline + Caps treated animals. Thus LPS can not modulate the time spend on capsaicin-induced discomfort behavior.
4. Discussion In the present study, we found that a severe immunological challenge influences the processing of intracranial trigeminal nociception. This conclusion is based on the finding that pre-treatment of the animals with 37.5 mg/kg LPS enhanced the number of cells in the TNC I,II that exhibit c-fos protein expression after intracisternally applied capsaicin.
3.6. C-fos expression 3.6.1. TNC As is shown in Fig. 4, LPS does not affect the numbers of cells expressing c-fos protein in the outer layers of the TNC (Vehicle: 10 ± 2, LPS 0.75 mg/kg: 10 ± 1, LPS 37.5 mg/kg: 17 ± 5). The Saline + Caps group however demonstrated an increased number of c-fos positive cells in the TNC I,II
4.1. LPS-induced sickness An enhanced loss of body weight in animals treated with both the low or high concentration of LPS was found in the
Table 2 Number of c-fos positive cells in the Area Postrema (AP) and different parts of the Nucleus of the Solitary Tract (NTS) in animals treated with i.p. saline (control, sal.), 0.75 mg/kg LPS (LPS(L)) or 37.5 mg/kg LPS (LPS(H)) injection 5 h prior to intracisternally infusion of vehicle (Control, Veh.) or capsaicin 250 nM (Caps) Control (n = 5) mean ± SEM Area Postrema Caudal NTS Medial Caudal NTS Lateral Rostral NTS Medial Rostral NTS Lateral
4 4 2 3 2
± ± ± ± ±
3 2 1 1 0
LPS(L) + Veh. (n = 6) mean ± SEM 14 51 7 6 4
*Significantly different from control (P , 0.05). **Significantly different from LPS (L) (P , 0.05).
± ± ± ± ±
3 13* 1* 1 1
LPS(H) + Veh. (n = 5) mean ± SEM 33 152 11 12 4
± ± ± ± ±
5*,** 1112*,** 212*,** 5 2
Sal. + Caps (n = 8) mean ± SEM 39 69 11 13 8
± ± ± ± ±
23* 38* 3* 4 2*
LPS(L) + Caps (n = 8) mean ± SEM 40 80 14 16 6
± ± ± ± ±
21 27 3 3 1
LPS(H) + Caps (n = 7) mean ± SEM 88 163 17 25 5
± ± ± ± ±
34 46 5 7 1
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current study. This enhanced weight loss caused by LPS may be induced by anorexia (Uehara et al., 1989; Pezeshki et al., 1996; Faggioni et al., 1997) or by increased energy consumption associated with fever (Long et al., 1990; Hua et al., 1996). As both LPS-induced anorexia and fever seem to be secondary to the production of cytokines, especially IL-1 (Long et al., 1990; Uehara et al., 1993; Hua et al., 1996; Pezeshki et al., 1996), the weight loss found in the present study may be considered indicative of a challenged, activated immune system. Although no difference in enhanced weight loss could be observed between animals treated with the low or high concentration LPS, other signs of an activated immune system do indicate differences in sickness severity between LPS(L) and LPS(H) treated animals. The pilo-erection and loss of posture shown by LPS(H) treated animals were not quantified but reduction of locomotor activity, which is a well known sickness behavior after LPS treatment (Yirmiya et al., 1994; Pezeshki et al., 1996), was quantified by measuring the time of immobilization shown by LPS treated rats during the infusion of vehicle solution. As capsaicin itself also induces immobilization behavior, effects of LPS on immobilization behavior can only be studied in animals that are not subject to capsaicin infusion. Control rats almost exclusively show exploring behavior during the 2 min of vehicle infusion. The novel environment of the cage that is used during infusion most likely induces this behavior. During i.c. infusion of vehicle only the LPS(H) concentration induced immobilization behavior and a significant reduction of exploration. This indicates that although the weight loss in the LPS(L) and LPS(H) treated animals is comparable, the LPS(H) treated animals do suffer more from the higher LPS concentration compared to the LPS(L) treated animals. The concentrations of LPS-type E. Coli 0.26:B6 used in the present experiments seem relatively high compared to other studies using LPS (Long et al., 1990; Hua et al., 1996; Pezeshki et al., 1996). However the lack of changes in immobilization behavior and the absence of pilo-erection in the LPS(L) treated animals shows that relatively high concentrations of this LPS-type are necessary to induce sickness behavior. 4.2. LPS sensitization of capsaicin sensitive afferents There are two findings in this study that point to LPS potentiation of capsaicin-induced intracranial trigeminal activation. First of all there is the above-mentioned enhancement of capsaicin-induced c-fos expression in the outer layers of the TNC by LPS(H). The TNC is the primary relay station for nociceptive trigeminal afferents (Henry et al., 1980) and especially cells in layer I and II of the TNC are termination sites of small unmyelinated C-fibers that react to nociceptive stimuli (Nozaki et al., 1992; Kaube et al., 1993). As c-fos is considered a marker for sensory neuronal activation (Hunt et al., 1987), the increased number of cells that express c-fos protein in the outer layers of the TNC
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indicates (1) that more nociceptive trigeminal afferents become activated or (2) that the synaptic transmission from first to second order neurons in the TNC is enhanced. Thus LPS enhances nociceptive trigeminal processing. A second finding from this study that points to potentiation of capsaicin-induced effects by LPS is that although LPS(L) alone did not induce immobilization behavior, this low dose could enhance the capsaicin-induced immobilization behavior. This effect of LPS(L) on immobilization behavior does suggest a sensitization of intracranial trigeminal afferents. An important issue in considering the effect of LPS(L) on capsaicin-induced immobilization behavior is that LPS(L) could not potentiate capsaicin-induced c-fos expression in the TNC I,II. Moreover, c-fos data point to hyposensitivity rather than hypersensitivity of the trigeminal afferents. This discrepancy between TNC I,II c-fos expression and immobilization behavior may be explained by the difference in temporal resolution of both parameters. As c-fos is an accumulation of cell activating events in the hours preceding perfusion, c-fos in the TNC I,II has little to no temporal resolution if it is considered as parameter of trigeminal nociception. Immobilization behavior however is measured acutely during the i.c. infusion of capsaicin and directly hereafter. The increase in capsaicin-induced immobilization behavior by LPS(L) is transient and may therefore not be reflected in TNC I,II c-fos expression. An alternative explanation for the discrepancy between cfos expression in the TNC I,II and immobilization behavior is that capsaicin may activate not only trigeminal pathways but also other afferent pathways. Two alternative pathways may especially be relevant in this model. Capsaicin is infused into the Cisterna Magna. This site is located near the Area Postrema, a brain region that contains capsaicin receptors (Szallasi et al., 1995). The Area Postrema projects heavily to the NTS (Ferguson and Lowes, 1994) which is the primary relay station for visceral afferent information and the NTS has pronounced ascending projection patterns throughout the brain (Ter Horst and Streefland, 1994). Capsaicin enhanced the c-fos expression of both the AP and caudomedial NTS, confirming that this pathway is activated. A second alternative pathway, also involving the NTS, that might be activated after intracisternal capsaicin infusion is mediated through vagal afferents. Vagal afferents are a possible target for intracisternally applied capsaicin because they innervate both the basilar artery and the dura mater (Keller et al., 1985a,b). Several branches of vagal afferents are sensitive for capsaicin (Lundberg and Saria, 1982; Holzer et al., 1994) and vagal afferents can contain Substance P (SP) (Gamse et al., 1979), a neuropeptide that, although debated in migraine (Diener, 1996; Goldstein et al., 1997), has been associated with nociception of the head (Moskowitz et al., 1979, 1984, Nakano et al., 1993). Also, like trigeminal afferents (Sugimoto et al., 1997; Henry et al., 1996), specific vagal afferent branches
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(Cadieux et al., 1986) and ganglia (Czyzyk-Krzeska et al., 1991) have been reported to show CGRP immunoreactivity and CGRP mRNA, respectively. CGRP in the venous outflow of the head has been found elevated in migraine patients during a migraine attack (Goadsby et al., 1990) and in nitroglycerin induced cluster headache (Fanciullacci et al., 1995), associating this neuropeptide with headache. Additionally the increased release of CGRP from rat tracheal perfusates after nociceptive treatment with capsaicin as described by Hua and colleagues (Hua et al., 1996) is thought to be derived from vagal afferents that innervate the trachea. Thus vagal afferents, like trigeminal afferents, are (1) localized in the meninges (2) contain neuropeptides that are associated with headache and (3) are reactive to capsaicin. As the primary termination site of vagal afferents in the brain is the NTS, counting the number of c-fos positive cells in the NTS, as was done in these experiments, could possibly elucidate whether capsaicin-induced activation of the NTS can be enhanced by LPS(L). This in turn might explain that LPS(L) enhances the capsaicin-induced immobilization behavior without affecting the c-fos expression in the TNC I,II. Capsaicin indeed does induce c-fos expression in the NTS, especially in the caudomedial part that receives input from general visceral afferents (opposite to the rostral part that predominantly receives gustatory information) (reviewed in Saper, 1995). However, as LPS(L) alone also induces c-fos expression in that part of the NTS, a potential enhancement of capsaicin-induced c-fos expression in the NTS by LPS(L) cannot be detected. 4.3. LPS-induced hyperalgesia Several explanations for increased sensitivity of nociceptive afferents after an immunological challenge have been put forward in literature. A neurocircuitry has been proposed to be involved in illness-induced hyperalgesia (Watkins et al., 1994a). The tail flick latency to radiant heat was tested in several conditions. Using this paradigm it was demonstrated that illness inducing agents produce hyperalgesia by initiating the production of cytokines. The cytokines in turn activate a pathway that subsequently involves the hepatic branch of the vagus, a circuitry in the brain involving the NTS and probably the nucleus raphe magnus and dorsal medial hypothalamus and a pathway in the dorsal funiculus of the spinal cord (Watkins et al., 1994a). Although hyperalgesia was tested 1 h after LPS administration instead of the 5 h used in the present investigation, evidence for involvement of this neurocircuitry in our experiments is found in the dose dependent increase of c-fos expression in the NTS after LPS administration. Besides affecting hepatic vagal nerves, cytokines produced after LPS administration also act directly within the brain. Several cytokines, including TNF-a and IL-1b are transported from blood to the brain by a saturable transport system (Banks et al., 1991). Intracerebroventricular (i.c.v.)
injection of IL-1b is able to induce anorexia (Plata-Salama´n et al., 1988; Uehara et al., 1989, 1993; Sonti et al., 1997) in rats. Also, in two different animal models (McHugh et al., 1994; Laviano et al., 1995) anorexia induced by a peripheral immunological challenge could be antagonized by central administration of an interleukin 1 receptor antagonist. The enhanced decrease in body weight induced by LPS treatments in the present study may indicate involvement of central IL-1 receptors. Interestingly, intracerebroventricular injection of IL-1b has been shown to induce hyperalgesia at the level of the trigeminal nucleus after stimulation of extracranial trigeminal afferents in rats (Oka et al., 1993). The neuronal substrate for this central action of IL-1b probably resides in the hypothalamus (Oka et al., 1993). Thus, cytokines might induce hyperalgesia in the present experiments by acting at peripheral vagal afferents or at central brain regions. A final site of action of cytokines that has to be considered is the effect that cytokines may have at the intracranial nociceptive afferents themselves. LPS, and more specifically the cytokines TNF-a and IL1b are able to increase the capsaicin-induced CGRP release from tracheal afferents (Hua et al., 1996). The immunological stimulation was performed in vivo but as the capsaicin stimulation was performed in vitro experiments (tracheal perfusates that were dissected from rats), it can be excluded that specific brain regions are involved in facilitating the capsaicin-induced CGRP release. More likely, the cytokines TNF-a and IL-1b act directly at the afferent nerve terminals. In conclusion, cytokines that are produced after LPS infusion might act at hepatic vagal afferents, central brain regions or nociceptive afferent nerve terminals to induce hyperalgesia. These different actions of cytokines do not exclude each other. Experiments are currently performed to elucidate which mechanism is most relevant for the hyperalgesic effects of LPS on intracranial trigeminal afferents. 4.4. Possible role of immune system-induced hyperalgesia in headache and migraine The most pronounced effect found in this study is that LPS(H) could potentiate the capsaicin-induced TNC I,II cfos expression. Although the 37.5 mg/kg LPS concentration is quite severe it strongly suggests that trigeminal afferents can become hyperalgesic after an immunological challenge. This may explain the reports that migraineous and nonmigraineous headache are of highest intensity after infection (Chabriat et al., 1997). Evidence in literature supports the relationship between infections and headache. Not only head and neck infections cause headache (Yoshikawa and Quinn, 1988) but also well known infections like influenza and HIV are linked to headache (Denning, 1988; Nicholson, 1992). Less well known infections like Japanese spotted fever and intrasellar infection are also associated by headache (Berger et al., 1986;
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Mahara, 1997). Headache after encephalitis, sinusitis or meningitis is well described (Wahlberg et al., 1989; Bross and Gordon, 1991; Rosenfeld and Rowley, 1994; Scelsa et al., 1995). It is clear from the above that different kind of infections can trigger headache. The present results suggest that the association between infections and headache may involve immune activation-induced hyperalgesia of nociceptive sensory nerves of the head. Several reports of altered immune system function in migraine patients have been put forward by Covelli and colleagues (Covelli et al., 1990a,b, 1991a,b). Increased spontaneous TNF-a release and a deficit of killing/phagocytosis of polymorphs and monocytes was found in migraine patients without aura compared to controls. Also a significant increase in T-lymphocyte subsets was found in migraine patients compared to healthy controls (Massari et al., 1994). It is suggested that these altered immune system functions may be responsible for the vascular, haemodynamic and pro-inflammatory symptoms associated with migraine (Covelli et al., 1991b). However no differences in TNF-a and IL-1 plasma levels could be found during and in-between attacks (van Hilten et al., 1991) in migraine patients suggesting that these cytokines are not involved in the initiation of the attack. Nevertheless, immunological dysfunction could make migraine patients more susceptible to migraine initiating events by causing hyperalgesia of nociceptive afferents of the head. This is supported by the present results. In conclusion, the number of cells expressing c-fos in the outer layers of the TNC after capsaicin treatment is increased by 37.5 mg/kg LPS. Thus trigeminal nociceptive processing can be enhanced by a severe immunological challenge. Immobilization behavior, induced by intracisternally applied capsaicin, can be enhanced by 0.75 mg/kg LPS. These hyperalgesic effects of LPS on capsaicin sensitive afferents are probably mediated by cytokines that act at peripheral vagal nerves, central brain regions or via direct actions of cytokines on capsaicin sensitive afferent nerve terminals. The hyperalgesic action of LPS found on trigeminal and possibly other capsaicin sensitive afferents of the head may explain the reports that headache can be triggered by different types of infections and that migraineous and non-migraineous headache is of highest intensity after an infection.
Acknowledgements The authors would like to thank Glaxo-Wellcome, Zeist, the Netherlands for their generous financial support.
References Banks, W.A., Ortiz, L., Plotkin, S.R. and Kastin, A.J., Human interleukin (IL) 1 alpha, murine IL-1 alpha and murine IL-1 beta are transported
189
from blood to brain in the mouse by a shared saturable mechanism, J. Pharmacol. Exp. Ther., 259 (1991) 988–996. Berger, S.A., Edberg, S.C. and David, G., Infectious disease in the sella turcica, Rev. Infect. Dis., 8 (1986) 745–755. Bross, J.E. and Gordon, G., Nocardial meningitis: case reports and review, Rev. Infect. Dis., 13 (1991) 160–165. Buzzi, M.G. and Moskowitz, M.A., The trigemino-vascular system and migraine, Pathol. Biol. (Paris), 40 (1992) 313–317. Cadieux, A., Springall, D.R., Mulderry, P.K., Rodrigo, J., Ghatei, M.A., Terenghi, G., Bloom, S.R. and Polak, J.M., Occurrence, distribution and ontogeny of CGRP immunoreactivity in the rat lower respiratory tract: effect of capsaicin treatment and surgical denervations, Neuroscience, 19 (1986) 605–627. Chabriat, H., Danchot, J., Michel, P., Joire, J.E. and Henry, P., Precipitating factors in migraineurs: a reappraisal in a national control-matched survey, Cephalalgia, 17 (1997) 318–319. Covelli, V., Maffione, A.B., Munno, I. and Jirillo, E., Alterations of nonspecific immunity in patients with common migraine, J. Clin. Lab. Anal., 4 (1990a) 9–15. Covelli, V., Munno, I., Pellegrino, N.M., Di, Venere A., Jirillo, E. and Buscaino, G.A., Exaggerated spontaneous release of tumor necrosis factor-alpha-cachectin in patients with migraine without aura, Acta Neurol. (Napoli), 45 (1990b) 257–263. Covelli, V., Massari, F., Fallacara, C., Munno, I., Pellegrino, N.M., Jirillo, E., Savastano, S., Ghiggi, M.R., Tommaselli, A.P. and Lombardi, G., Increased spontaneous release of tumor necrosis factor- alpha/cachectin in headache patients. A possible correlation with plasma endotoxin and hypothalamic-pituitary-adrenal axis, Int. J. Neurosci., 61 (1991a) 53– 60. Covelli, V., Munno, I., Pellegrino, N.M., Altamura, M., Decandia, P., Marcuccio, C., Di Venere, A. and Jirillo, E., Are TNF-alpha and IL-1 beta relevant in the pathogenesis of migraine without aura?, Acta Neurol. (Napoli), 13 (1991b) 205–211. Cunha, F.Q., Poole, S., Lorenzetti, B.B. and Ferreira, S.H., The pivotal role of tumour necrosis factor alpha in the development of inflammatory hyperalgesia, Br. J. Pharmacol., 107 (1992) 660–664. Czyzyk-Krzeska, M.F., Bayliss, D.A., Seroogy, K.B. and Millhorn, D.E., Gene expression for peptides in neurons of the petrosal and nodose ganglia in rat, Exp. Brain Res., 83 (1991) 411–418. Denning, D.W., The neurological features of acute HIV infection, Biomed. Pharmacother., 42 (1988) 11–14. Diener, H.C., Substance-P antagonist RPR100893–201 is not effective in human migraine attacks. In: J. Olesen and P. Tfelt-Hansen (Eds.), Proceedings of the VIth International Headache Seminar, Lippincot-Raven, New York, 1996. Faggioni, R., Fuller, J., Moser, A., Feingold, K.R. and Grunfeld, C., LPSinduced anorexia in leptin-deficient (ob/ob) and leptin receptor- deficient (db/db) mice, Am. J. Physiol., 42 (1997) R181–R186. Fanciullacci, M., Allessandri, M., Figini, M., Geppetti, P. and Michelacci, S., Increase in plasm calcitonin gene-related peptide from the extracerebral circulation during nitroglycerin-induced cluster headache attack, Pain, 60 (1995) 119–123. Ferguson, A.V. and Lowes, V.L., Functional neural connections of the Area Postrema. In: I.R.A. Barraca (Ed.), Nucleus of the solitary tract, CRC Press, Boca Raton, 1994, pp. 147–157. Ferreira, S.H., Lorenzetti, B.B., Bristow, A.F. and Poole, S., Interleukin-1 beta as a potent hyperalgesic agent antagonized by a tripeptide analogue, Nature, 334 (1988) 698–700. Gamse, R., Lembeck, F. and Cuello, A.C., Substance P in the vagus nerve. Immunochemical and immunohistochemical evidence for axoplasmic transport, Naunyn Schmiedebergs Arch. Pharmacol., 306 (1979) 37– 44. Goadsby, P.J., Edvinsson, L. and Ekman, R., Vasoactive peptide release in the extracerebral circulation of humans during migraine headache, Ann. Neurol., 28 (1990) 183–187. Goadsby, P.J. and Edvinsson, L., The trigeminovascular system and migraine: studies characterizing cerebrovascular and neuropeptide
190
R.H.A. Kemper et al. / Pain 78 (1998) 181–190
changes seen in humans and cats, Ann. Neurol., 33 (1993) 48– 56. Goldstein, D.J., Wang, O., Saper, J.R., Stoltz, R., Silberstein, S.D. and Mathew, N.T., Ineffectiveness of neurokinin-1 antagonists in acute migraine: a crossover study, Cephalalgia, 17 (1997) 785–790. Henry, J.L., Sessle, B.J., Lucier, G.E. and Hu, J.W., Effects of substance P on nociceptive and non-nociceptive trigeminal brain stem neurons, Pain, 8 (1980) 33–45. Henry, M.A., Johnson, L.R., Nousek-Goebl, N. and Westrum, L.E., Light microscopic localization of calcitonin gene-related peptide in the normal feline trigeminal system and following retrogasserian rhizotomy, J. Comp. Neurol., 365 (1996) 526–540. Herbert, M.K. and Holzer, P., Interleukin-1 beta enhances capsaicininduced neurogenic vasodilatation in the rat skin, Br. J. Pharmacol., 111 (1994) 681–686. Holzer, H.H., Turkelson, C.M., Solomon, T.E. and Raybould, H.E., Intestinal lipid inhibits gastric emptying via CCK and a vagal capsaicinsensitive afferent pathway in rats, Am. J. Physiol., 267 (1994) G625– 629. Hua, X.Y., Chen, P., Fox, A. and Myers, R.R., Involvement of cytokines in lipopolysaccharide-induced facilitation of CGRP release from capsaicin-sensitive nerves in the trachea: studies with interleukin-1beta and tumor necrosis factor-alpha, J. Neurosci., 16 (1996) 4742–4748. Hunt, S.P., Pini, A. and Evan, G., Induction of c-fos-like protein in spinal cord neurons following sensory stimulation, Nature, 328 (1987) 632– 634. Kaube, H., Keay, K.A., Hoskin, K.L., Bandler, R. and Goadsby, P.J., Expression of c-Fos-like immunoreactivity in the caudal medulla and upper cervical spinal cord following stimulation of the superior sagittal sinus in the cat, Brain Res., 629 (1993) 95–102. Keller, J.T., Beduk, A. and Saunders, M.C., Origin of fibers innervating the basilar artery of the cat, Neurosci. Lett., 58 (1985a) 263–268. Keller, J.T., Saunders, M.C., Beduk, A. and Jollis, J.G., Innervation of the posterior fossa dura of the cat, Brain Res. Bull., 14 (1985b) 97–102. Kemper, R.H., Meijler, W.J. and TerHorst, G.J., Trigeminovascular stimulation in conscious rats, NeuroReport, 8 (1997) 1123–1126. Laviano, A., Renvyle, T., Meguid, M.M., Yang, Z.J., Cangiano, C. and Rossi, F.F., Relationship between interleukin-1 and cancer anorexia, Nutrition, 11 (1995) 680–683. Long, N.C., Otterness, I., Kunkel, S.L., Vander, A.J. and Kluger, M.J., Roles of interleukin 1 beta and tumor necrosis factor in lipopolysaccharide fever in rats, Am. J. Physiol., 259 (1990) R724–728. Lundberg, J.M. and Saria, A., Capsaicin-sensitive vagal neurons involved in control of vascular permeability in rat trachea, Acta Physiol. Scand., 115 (1982) 521–523. Mahara, F., Japanese spotted fever: report of 31 cases and review of the literature, Emerg. Infect. Dis., 3 (1997) 105–111. Massari, F., D’Andrea, L., Cervo, M.A., Serra, F.P., Covelli, V. and Buscaino, G.A., Quantitative and qualitative modifications of lymphocyte subsets after sublingual administration of isosorbide dinitrate in migraineurs. Preliminary report, Acta Neurol. (Napoli), 16 (1994) 11– 18. McHugh, K.J., Collins, S.M. and Weingarten, H.P., Central interleukin-1 receptors contribute to suppression of feeding after acute colitis in the rat, Am. J. Physiol., 266 (1994) R1659–R1663. Morrison, D.C., Endotoxin and disease mechanisms, Annu. Rev. Med., 38 (1987) 417–432. Moskowitz, M.A., Reinhard, J.F. Jr., Romero, J., Melamed, E. and Pettibone, D.J., Neurotransmitters and the fifth cranial nerve: is there a relation to the headache phase of migraine? Lancet, 2 (1979) 883– 885. Moskowitz, M.A., The neurobiology of vascular head pain, Ann. Neurol., 16 (1984) 157–168. Nakano, T., Shimomura, T., Takahashi, K. and Ikawa, S., Platelet substance P and 5-hydroxytryptamine in migraine and tension-type headache, Headache, 33 (1993) 528–532. Nozaki, K., Moskowitz, M.A. and Boccalini, P., CP-93,129, sumatriptan,
dihydroergotamine block c-fos expression within rat trigeminal nucleus caudalis caused by chemical stimulation of the meninges, Br. J. Pharmacol., 106 (1992) 409–415. Nicholson, K.G., Clinical features of influenza, Semin. Respir. Infect., 7 (1992) 26–34. Oka, T., Aou, S. and Hori, T., Intracerebroventricular injection of interleukin-1 beta induces hyperalgesia in rats, Brain Res., 624 (1993) 61– 68. Pezeshki, G., Pohl, T. and Schobitz, B., Corticosterone controls interleukin-1 beta expression and sickness behavior in the rat, J Neuroendocrinol., 8 (2) (1996) 129–135. Paxinos, G. and Watson, C., The rat brain in stereotactic coordinates. Compact third edition, (1997). Plata-Salama´n, C.R., Oomura, Y. and Kai, Y., Tumor necrosis factor and interleukin-1 beta: suppression of food intake by direct action in the central nervous system, Brain Res., 448 (1988) 106–114. Rosenfeld, E.A. and Rowley, A.H., Infectious intracranial complications of sinusitis, other than meningitis, in children: a 12-year review, Clin. Infect. Dis., 18 (1994) 750–754. Saper, C.B., Central autonomic system. In: G. Paxinos (Ed.), The rat nervous system; second edition, Academic Press, San Diego, 1995, pp. 107–135. Scelsa, S.N., Lipton, R.B., Sander, H. and Herskovitz, S., Headache characteristics in hospitalized patients with Lyme disease, Headache, 35 (1995) 125–130. Sonti, G., Flynn, M.C. and Plata-Salama´n, C.R., Interleukin-1 (IL-1) receptor type I mediates anorexia but not adipsia induced by centrally administered IL-1beta, Physiol. Behav., 62 (1997) 1179–1183. Sugimoto, T., Fujiyoshi, Y., Xiao, C., He, Y.F. and Ichikawa, H., Central projection of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive trigeminal primary neurons in the rat, J. Comp. Neurol., 378 (1997) 425–442. Szallasi, A., Nilsson, S., Farkas, S.T., Blumberg, P.M., Hokfelt, T. and Lundberg, J.M., Vanilloid (capsaicin) receptors in the rat: distribution in the brain, regional differences in the spinal cord, axonal transport to the periphery, and depletion by systemic vanilloid treatment, Brain Res., 703 (1995) 175–183. Ter Horst, G.J. and Streefland, C., Ascending projections of the solitary tract nucleus, In: I.R.A. Barraca (Ed.), Nucleus of the solitary tract, CRC Press, Boca Raton, 1994, pp. 93–103. Uehara, A., Sekiya, C., Takasugi, Y., Namiki, M. and Arimura, A., Anorexia induced by interleukin 1: involvement of corticotropin-releasing factor, Am. J. Physiol., 257 (1989) R613–R617. Uehara, Y., Shimizu, H., Sato, N., Mura, Y.S. and Mori, M., The dipeptide Lys-Pro attenuates interleukin-1 beta-induced anorexia, Peptides, 14 (1993) 175–178. van Hilten, J.J., Ferrari, M.D., Van der Meer, J.W., Gijsman, H.J. and Looij, B.J. Jr., Plasma interleukin-1, tumour necrosis factor and hypothalamic- pituitary-adrenal axis responses during migraine attacks, Cephalalgia, 11 (1991) 65–67. Wahlberg, P., Saikku, P. and Brummer-Korvenkontio, M., Tick-borne viral encephalitis in Finland. The clinical features of Kumlinge disease during 1959–1987, J. Intern. Med., 225 (1989) 173–177. Watkins, L.R., Wiertelak, E.P., Goehler, L.E., Mooney-Heiberger K., Martinez, J., Furness, L., Smith, K.P. and Maier, S.F., Neurocircuitry of illness-induced hyperalgesia, Brain Res., 639 (1994a) 283–299. Watkins, L.R., Wiertelak, E.P., Goehler, L.E., Smith, K.P., Martin, D., and Maier, S.F., Characterization of cytokine-induced hyperalgesia, Brain Res 654 (1994b) 15–26. Yirmiya, R., Rosen, H., Donchin, O. and Ovadia, H., Behavioral effects of lipopolysaccharide in rats: involvement of endogenous opioids, Brain Res., 648 (1994) 80–86. Yoshikawa, T.T. and Quinn, W., The aching head. Intracranial suppuration due to head and neck infections, Infect. Dis. Clin. North. Am., 2 (1988) 265–277. Zimmermann, M., Ethical guidelines for investigations of experimental pain in conscious animals [editorial], Pain, 16 (1983) 109–110.
