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Multiple neural mechanisms of fever
Autonomic Neuroscience: Basic and Clinical 85 (2000) 78–82 www.elsevier.com / locate / autneu
Short communication
Multiple neural mechanisms of fever a ´ Szekely ´ Miklos , Marta Balasko´ a , Vladimir A. Kulchitsky b , Christopher T. Simons b , b,c b,c , Andrei I. Ivanov , Andrej A. Romanovsky * a ´ , H7643 Pecs ´ , Hungary Department of Pathophysiology, University Medical School Pecs Thermoregulation Laboratory, Legacy Holladay Park Medical Center, Portland, OR 97140, USA c Trauma Research, St. Joseph’ s Hospital and Medical Center, 350 West Thomas Road, Phoenix, AZ 85013, USA b
Abstract In rats, fevers induced by moderate-to-high doses of intravenous lipopolysaccharide consist of three phases (phases 1, 2 and 3) with body temperature peaks at |1, 2, and 5 h postinjection, respectively. In this study, the effects of bilateral truncal subdiaphragmatic vagotomy and intraperitoneal capsaicin desensitization on febrile phases 1–3 were assessed in adult Wistar rats. Surgical vagotomy was performed |30 d before the experiment; this procedure interrupts both afferent and efferent vagal fibers. Capsaicin was administered intraperitoneally in two consecutive injections (2 and 3 mg / kg, 3 h apart) 1 week prior to the experiment; this procedure desensitizes afferent fibers, primarily within the abdominal cavity, and does not lead to the known thermal effects of systemic capsaicin desensitization. At a neutral ambient temperature, the rats were given Escherichia coli lipopolysaccharide (10 mg / kg) through a preimplanted jugular catheter, and their colonic temperature wes measured by thermocouples for 7 h. The control rats exhibited the typical triphasic febrile responses. Confirming our earlier studies, subdiaphragmatic vagotomy did not affect phases 1 and 2; it did, however, result in a 2.5-fold reduction of phase 3. Capsaicin desensitization modified the febrile response differently: phases 2 and 3 were unaffected, but phase 1 disappeared. We suggest that neural afferent fibers (nonvagal but perhaps vagal as well) play an important role in the early febrile response (phase 1) by transducing peripheral pyrogenic signals to the brain. We also suggest that vagal efferent fibers are likely to participate in the later febrile response (phase 3) via an unknown mechanism. 2000 Elsevier Science B.V. All rights reserved. Keywords: Febrile phases; Endotoxin; Lipopolysaccharide; Vagus nerve; Subdiaphragmatic vagotomy; Capsaicin; Intra-abdominal neural afferents
The febrile response of rats to moderate-to-high doses of intravenous (i.v.) lipopolysaccharide (LPS) is polyphasic. The first three phases (phases 1, 2 and 3) of this response have been identified and are characterized by body (colonic) temperature (T c ) peaks at |1, 2, and 5 h postinjection, respectively (Romanovsky et al., 1998). The short latency of phase 1 suggests that it is mediated neurally. Afferent vagal fibers, perhaps of the hepatic branch, have been proposed as the anatomical substrate of such mediation (Maier et al., 1998; Goehler et al., 2000; Romanovsky et al., 2000b). Reportedly, truncal subdiaphragmatic vagotomy (Sehic and Blatteis, 1996; Romanovsky et al., 1997a,c), selective transection of the hepatic vagal branch (Simons et al., 1998), and capsaicin ´ desensitization of abdominal neural afferents (Szekely et *Corresponding author. Trauma Research, St. Joseph’s Hospital and Medical Center, 350 West Thomas Road, Phoenix, AZ 85013, USA. Tel.: 11-602-406-5059; fax: 11-602-406-4113. E-mail address: [email protected] (A.A. Romanovsky).
al., 1995) all attenuate the febrile response to i.v. LPS. However, the effects of vagotomy, whether surgical or ‘‘chemical’’ (capsaicin desensitization), on phases 1 and 2 of the polyphasic febrile response to i.v. or intraarterial LPS remain controversial (Sehic and Blatteis, 1996; Romanovsky et al., 1997c; Caldwell et al., 1999), whereas the effect of vagotomy on the recently discovered phase 3 has not been studied yet. The aim of the present study was to determine how febrile phases 1, 2, and 3 are affected by truncal subdiaphragmatic vagotomy (experiment 1) and capsaicin desensitization (experiment 2). The latter procedure affects only afferent C- and Ad-fibers, both vagal and nonvagal (Holzer, 1988); the former procedure interrupts both afferent and efferent vagal fibers. Importantly, truncal vagotomy does not affect most sympathetic and adventitial fibers that course with the vagus, because these fibers are abundant at the branch level but scarce in the trunks (Prechtl and Powley, 1990). Experiment 1 was conducted in 60 adult male Wistar rats (B and K Universal, Kent, WA, USA). The animals
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´ et al. / Autonomic Neuroscience: Basic and Clinical 85 (2000) 78 – 82 M. Szekely
were caged singly at an ambient temperature (T a ) of |228C. Food and water were available ad libitum; the vagotomized animals were given reinforced food. As in the past (Romanovsky et al., 1997a), the rats were subdiaphragmatically vagotomized (n528) or sham vagotomized (n532) |30 d before the experiment and had a catheter implanted in the right jugular vein 3 d before the experiment. The rats were extensively habituated to the experimental conditions. On the day of the test, each rat was instrumented with a colonic thermocouple connected to a data acquisitor (Model TCA-AI-24; Dianachart, Rockaway, NJ, USA) and personal computer. The animal was then placed in an individual stock and transferred to a climatic chamber (Forma Scientific, Marietta, OH, USA) set to a neutral T a of 30.08C and 50.0% relative humidity. The exteriorized portion of the i.v. catheter was passed through a wall port and connected to a syringe. After a 1-h stabilization period, measurements were begun, and T c was recorded every 2 min from 1 h before to 7 h after the i.v. injection of Escherichia coli LPS (O111:B4; Sigma, St. Louis, MO, USA) in a dose of 10 mg / kg in pyrogen-free saline (PFS; 1 ml / kg) or PFS alone. The effectiveness of surgical vagotomy was verified in six vagotomized and six sham-operated rats randomly selected from the study population. A few days after the experiment, these 12 animals were food-deprived for 24 h and thereafter euthanized with a barbiturate injection. The stomachs together with their contents were removed and weighed. Subdiaphragmatic vagotomy is known to cause gastric dilatation and inhibit the evacuatory function of the stomach, leading to increased stomach mass (Kraly et al., 1986; Romanovsky et al., 1997a). Experiment 2 was conducted in 31 adult Wistar rats ´ ´ (University Medical School Pecs, Pecs, Hungary). The animals were adapted to cold by being caged singly in standard ‘‘shoeboxes’’ with excessive woodchip bedding (to absorb moisture and allow nest building) at a T a of |58C for at least 30 d. Cold adaptation was used to increase the magnitude of the first febrile phase, the major focus of this experiment. The first phase is relatively low in nonadapted rats, but it is much higher in cold-adapted ´ rats (Szekely, unpublished data) that have been shown to develop robust thermogenic and hyperthermic responses ´ (Szekely and Mercer, 1999). One week before the experiment, the intra-abdominal neural afferents were desensitized in 14 rats by two consecutive intraperitoneal (i.p.) injections of capsaicin (8-methyl-N-vanillyl-6nonenamide; Sigma), 2 and 3 mg / kg, 3 h apart. A stock solution of capsaicin in absolute ethanol was freshly diluted with PFS to final concentrations of capsaicin and ethanol of 1 mg / ml and 10%, respectively. Low-dose i.p. desensitization was used because it causes transient (3–5 weeks) neural damage that is limited primarily to the abdominal cavity and does not lead to the known systemic ´ effects of desensitization with high doses (Szekely and Romanovsky, 1998). Sham animals (n517) received no
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capsaicin. The rats had a catheter implanted in the right jugular vein 3 d before the experiment. As in the past ´ (Szelenyi et al., 1994), each rat was instrumented with a colonic thermocouple and placed into a brass open-circuit metabolic chamber, which was immersed in a water bath. The chamber was maintained at 25.08C (a neutral T a for ´ cold-adapted rats; see Szekely and Mercer, 1999). The exteriorized portion of the i.v. catheter was passed through a wall port and connected to a syringe. After a 1-h stabilization period, the measurements were begun, and T c was recorded from 1 h before to 7 h after the i.v. injection of Escherichia coli LPS (O111:B4; 10 mg / kg) in PFS (1 ml / kg) or PFS alone. The effectiveness of capsaicin desensitization was verified in eight capsaicin-treated and 12 control rats randomly selected from the study population. A few days after the experiment, these 20 animals were food-deprived for 24 h and offered an excessive amount of standard rat chow thereafter. The animals were weighed twice, viz. immediately before and 150 min after food presentation. The difference in the body mass was calculated, expressed as a percentage of the initial body mass, and considered an index of the amount of food consumed over the 150 min. Neural (primarily vagal) afferents partially mediate satiety (Schwartz et al., 1999), and increased food consumption (satiety impairment) was expected in the capsaicin-treated animals. In both experiments, we calculated the fever index for each phase of the polyphasic febrile response as a time integral of the deviation of T c from its level at time 0 (LPS or PFS injection). The latter deviation was integrated over 30–70 min postinjection (phase 1), 70–180 min (phase 2), or 180–420 min (phase 3). The integration times were determined in advance, based on our previous data (Romanovsky et al., 1998). The fever indices were compared using Student’s unpaired t-test. This test was also used to compare stomach masses and food intakes for verification of the effectiveness of the vagotomy and capsaicin-desensitization procedures, respectively. In both experiments, all rats responded to i.v. LPS (but not PFS) with a polyphasic fever. The major results of experiment 1 are shown in Fig. 1. Compared to the sham-operated controls, the febrile response of the vagotomized rats had an identical phase 1 (7.964.2 min38C vs. 8.262.8 min38C; P55310 21 ), a similar phase 2 (60.4618.0 min38C vs. 74.1611.2 min38C; P523 10 21 ), and, on average, a |2.5-fold attenuated phase 3 (85.5653.1 min38C vs. 189.7639.5 min38C; P553 10 22 ). However, phase 3 in the vagotomized rats varied substantially: in the different runs of this experiment, it was normal, attenuated, or completely abolished compared to shams in the same subsets. The stomach mass of the vagotomized animals (3.860.4%) was |2.5-times higher than that of the shams (1.660.1%; P52310 24 ), thus confirming the effectiveness of surgery. The major results of experiment 2 are shown in Fig. 2. Compared to the controls, the capsaicin-desensitized rats
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´ et al. / Autonomic Neuroscience: Basic and Clinical 85 (2000) 78 – 82 M. Szekely
Fig. 1. The thermal response of the subdiaphragmatically vagotomized and sham-operated rats to an intravenous injection (arrow) of lipopolysaccharide (LPS, 10 mg / kg; upper panel) or pyrogen-free saline (PFS, 1.0 ml / kg; lower panel). The initial (time 0) colonic temperatures of the vagotomized rats were 38.460.18C (LPS) and 37.960.18C (PFS). The initial colonic temperatures of the shams were 38.360.18C (LPS) and 37.960.18C (PFS).
exhibited identical phase 2 (101.8618.3 min38C vs. 102.1623.2 min38C; P55310 21 ) and phase 3 (276.9663.4 min38C vs. 276.3644.4 min38C; P553 10 21 ). However, phase 1 of their febrile response was completely abolished (5.363.8 min38C vs. 24.766.9 min38C in the controls; P58310 23 ). This febrile pattern (no or low phase 1 – normal phase 2 – normal phase 3) was consistent in all capsaicin-pretreated animals. The food consumption (increase in body mass over 150 min of postdeprivational feeding) of the capsaicin-desensitized animals (7.660.4%) was significantly higher than that of
Fig. 2. The thermal response of the capsaicin-pretreated and control cold-acclimated rats to an intravenous injection (arrow) of lipopolysaccharide (LPS, 10 mg / kg; upper panel) or pyrogen-free saline (PFS, 1.0 ml / kg; lower panel). The initial (time 0) colonic temperatures of the capsaicin-pretreated rats were 37.660.28C (LPS) and 37.760.18C (PFS). The initial colonic temperatures of the controls were 37.460.28C (LPS) and 37.560.28C (PFS).
the shams (5.460.5%; P52310 23 ), thus independently confirming the effectiveness of desensitization. Confirming our earlier speculations (Romanovsky et al., 1996), this study demonstrates that different febrile phases are mediated differently. In the present experiments, phase 1 of the polyphasic febrile response to a moderate i.v. dose of LPS was unaffected by total subdiaphragmatic vagotomy, but it was abolished by capsaicin-induced damage of intra-abdominal afferent fibers. This suggests that neural afferents are involved in the genesis of phase 1, but that nonvagal chemosensitive fibers are more important than vagal. Together with our earlier findings (Romanovsky et al., 1997a,c), the present results also indicate that phase 1 of the polyphasic febrile response to moderate doses of LPS differs from the monophasic febrile response to very low, near-threshold doses. The latter is attenuated by subdiaphragmatic vagotomy (Romanovsky et al., 1997a,c), whereas the former is unaffected (Romanovsky et al., 1997c). The relationship between monophasic fever and phase 1 of polyphasic fever is currently unclear. In the present study, phase 2 of the polyphasic LPS fever was affected neither by total truncal subdiaphragmatic vagotomy nor by i.p. capsaicin pretreatment. This observation confirms our earlier data (Romanovsky et al., 1997c) and contradicts the data of Sehic and Blatteis (1996). Although interspecies differences (rats vs. guinea pigs; see, e.g., Metz and Forssmann, 1980) may underlie this contradiction, the potential nonspecific consequences / complications of subdiaphragmatic vagotomy (Alvarez, 1948; Kraly et al., 1986), including thermoeffector insufficiency (Lin and Chern, 1985), should also be considered. In our studies, the animals were maintained on a reinforced diet after surgery; the importance of this precaution has been emphasized repeatedly (Andrews et al., 1985; Romanovsky et al., 1997a). Moreover, we have directly demonstrated good health (Romanovsky et al., 1997a) and uncompromised thermoeffector responses in these rats (Romanovsky et al., 1997b; Sugimoto et al., 1999); such controls have often been neglected by others. In sum, based on the present experiments and our earlier studies (Romanovsky et al., 1997c), we are confident that phase 2 of the polyphasic febrile response is unaffected by vagotomy, whether surgical or chemical, at least in the rat. We suggest that either intra-abdominal nerve fibers (whether afferent vagal, efferent vagal, or afferent nonvagal) are uninvolved in the genesis of phase 2 or that extra-abdominal neural and / or humoral mechanisms readily compensate for the functional inactivity of intra-abdominal fibers in vagotomized and capsaicin-desensitized animals. Recently, we have identified phase 3 of the polyphasic febrile response of rats to i.v. LPS (Romanovsky et al., 1998). The genesis of this phase remains unclear. The present study showed that phase 3 of LPS fever was unaffected by capsaicin desensitization of abdominal neural afferents but was attenuated (to various degrees) by total subdiaphragmatic vagotomy. These findings suggest that
´ et al. / Autonomic Neuroscience: Basic and Clinical 85 (2000) 78 – 82 M. Szekely
capsaicin-insensitive vagal fibers, presumably efferents, are involved in the genesis of the febrile phase 3, and that this involvement is strongly modulated by unknown factors that were uncontrolled in the present experiments. Interestingly, the involvement of efferent vagal fibers in i.p. LPS-induced fever (Romanovsky et al., 2000a) and i.v. LPS-induced shock (Borovikova et al., 2000) has recently been demonstrated. We conclude that the three phases of the polyphasic febrile response of rats to i.v. LPS are differentially sensitive to vagotomy and, therefore, likely to be mediated differently. Phase 1 is abolished by capsaicin desensitization of intra-abdominal neural afferents but unaffected by truncal subdiaphragmatic vagotomy. Phase 2 is unchanged in both surgically vagotomized and capsaicin-desensitized rats. Phase 3 is unaffected by i.p. capsaicin pretreatment but attenuated by subdiaphragmatic vagotomy. We speculate that some intra-abdominal neural afferents are involved in the genesis of phase 1, and that nonvagal afferent fibers are more important for this phase than vagal afferents. We further suggest that intra-abdominal nerve fibers are uninvolved in the genesis of phase 2 or that other mechanisms readily compensate for their functional inactivity in vagotomized and capsaicin-desensitized animals. Finally, we propose that vagal efferents (but not vagal or nonvagal afferents) are involved in the genesis of the febrile phase 3, and that this involvement is strongly modulated by as of yet unidentified factors. The question ‘‘Is the vagus nerve involved in the febrile response?’’ is probably too general to be answered correctly. In the case of i.v. LPS-induced fever in rats, this question should be rephrased as follows: ‘‘Which type of neural fibers (afferent or efferent; vagal or nonvagal) is involved in each febrile phase?’’ Together with other papers in this issue, the present results suggest that multiple neural mechanisms underlie the fever response.
Acknowledgements The authors thank Dr. S. Kick and Ms. D. Mutchler for editing the manuscript. This study was supported in part by grants and donations from the National Institutes of Health (1 RO1 NS41233-01), Oregon Health Sciences Foundation (Medical Research Foundation of Oregon), Collins Medical Trust, Good Samaritan Foundation, Bayer AG, and Dr. Temple Fay Memorial Account to A.A.R. and from the Hungarian Scientific Research Grant Agency (OTKA T026511) to M.S. V.A.K. was on leave from the Institute of Physiology, Minsk, Belarus.
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Andrews, P.L., Rothwell, N.J., Stock, M.J., 1985. Effects of subdiaphragmatic vagotomy on energy balance and thermogenesis in the rat. J. Physiol. (Lond.) 362, 1–12. Borovikova, L.V., Ivanova, S., Zhang, M., Yang, H., Botchkina, G.L., Watkins, L.R., Wang, H., Abumrad, N., Eaton, J.W., Tracey, K.J., 2000. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 405, 458–462. Caldwell, Jr. F.T., Graves, D.B., Wallace, B.H., 1999. Humoral versus neural pathways for fever production in rats after administration of lipopolysaccharide. J. Trauma 47, 120–129. Goehler, L.E., Gaykema, R.P.A., Hansen, M.K., Andreson, K., Maier, S.F., Watkins, L.R., 2000. Vagal immune-to-brain communication: a visceral chemosensory pathway. Auton. Neurosci. 85, 49–59. Holzer, P., 1988. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 24, 739–768. Lin, M.T., Chern, Y.F., 1985. Effects of subdiaphragmatic vagotomy on thermoregulatory responses of rats to different ambient temperatures. Exp. Neurol. 88, 467–470. Kraly, F.S., Jerome, C., Smith, G.P., 1986. Specific postoperative syndromes after total and selective vagotomies in rats. Appetite 7, 1–17. Maier, S.F., Goehler, L.E., Fleshner, M., Watkins, L.R., 1998. The role of the vagus nerve in cytokine-to-brain communication. Ann. NY Acad. Sci. 840, 289–300. Metz, W., Forssmann, W.G., 1980. Innervation of the liver in guinea pig and rat. Anat. Embryol. 160, 239–252. Prechtl, J.C., Powley, T.L., 1990. The fiber composition of the abdominal vagus in the rat. Anat. Embryol. 181, 101–115. Romanovsky, A.A., Kulchitsky, V.A., Akulich, N.V., Koulchitsky, S.V., Simons, C.T., Sessler, D.I., Gourine, V.N., 1996. First and second phases of biphasic fever: two sequential stages of the sickness syndrome? Am. J. Physiol. 271, R244–R253. Romanovsky, A.A., Kulchitsky, V.A., Simons, C.T., Sugimoto, N., ´ Szekely, M., 1997a. Febrile responsiveness of vagotomized rats is suppressed even in the absence of malnutrition. Am. J. Physiol. 273, R777–R783. Romanovsky, A.A., Kulchitsky, V.A., Simons, C.T., Sugimoto, N., ´ Szekely, M., 1997b. Cold defense mechanisms in vagotomized rats. Am. J. Physiol. 273, R784–R789. ´ Romanovsky, A.A., Simons, C.T., Szekely, M., Kulchitsky, V.A., 1997c. The vagus nerve in the thermoregulatory response to systemic inflammation. Am. J. Physiol. 273, R407–R413. Romanovsky, A.A., Simons, C.T., Kulchitsky, V.A., 1998. ‘‘Biphasic’’ fevers often consist of more than two phases. Am. J. Physiol. 275, R323–R321. Romanovsky, A.A., Ivanov, A.I., Berthoud, H.-R., Kulchitsky, V.A., 2000a. Are vagal efferents involved in the fever response to intraperitoneal lipopolysaccharide? J. Therm. Biol. 25, 65–70. ´ Romanovsky, A.A., Ivanov, A.I., Szekely, M., 2000b. Neural route of pyrogen signaling to the brain. Clin. Infect. Dis. 31 (Suppl. 5), 162–167. Schwartz, G.J., Salorio, C.F., Skoglund, C., Moran, T.H., 1999. Gut vagal afferent lesions increase meal size but do not block gastric preloadinduced feeding suppression. Am. J. Physiol. 276, R1623–R1629. Sehic, E., Blatteis, C.M., 1996. Blockade of lipopolysaccharide-induced fever by subdiaphragmatic vagotomy in guinea pigs. Brain Res. 726, 160–166. ´ Simons, C.T., Kulchitsky, V.A., Sugimoto, N., Homer, L.D., Szekely, M., Romanovsky, A.A., 1998. Signaling the brain in systemic inflammation: which vagal branch is involved in fever genesis? Am. J. Physiol. 275, R63–R68. Sugimoto, N., Simons, C.T., Romanovsky, A.A., 1999. Vagotomy does not affect thermal responsiveness to intrabrain prostaglandin E 2 and cholecystokinin octapeptide. Brain Res. 844, 157–163. ´ Szekely, M., Mercer, J.B., 1999. Thermosensitivity changes in coldadapted rats. J. Therm. Biol. 24, 369–371.
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´ Szekely, M., Romanovsky, A.A., 1998. Thermoregulatory reactions of capsaicin-pretreated rats. FASEB J. 11, A528. ´ ´ M., Romanovsky, A.A., 1995. Capsaicin-sensitive Szekely, M., Balasko, ¨ neural afferents in fever pathogenesis. Pflugers Arch. 440 (Suppl.), R64.
´ ´ L., Szekely, ´ Szelenyi, Z., Bartho, M., Romanovsky, A.A., 1994. Cholecystokinin octapeptide (CCK-8) injected into a cerebral ventricle induces a fever-like thermoregulatory response mediated by type B CCK-receptors in the rat. Brain Res. 638, 69–77.
Short communication
Multiple neural mechanisms of fever a ´ Szekely ´ Miklos , Marta Balasko´ a , Vladimir A. Kulchitsky b , Christopher T. Simons b , b,c b,c , Andrei I. Ivanov , Andrej A. Romanovsky * a ´ , H7643 Pecs ´ , Hungary Department of Pathophysiology, University Medical School Pecs Thermoregulation Laboratory, Legacy Holladay Park Medical Center, Portland, OR 97140, USA c Trauma Research, St. Joseph’ s Hospital and Medical Center, 350 West Thomas Road, Phoenix, AZ 85013, USA b
Abstract In rats, fevers induced by moderate-to-high doses of intravenous lipopolysaccharide consist of three phases (phases 1, 2 and 3) with body temperature peaks at |1, 2, and 5 h postinjection, respectively. In this study, the effects of bilateral truncal subdiaphragmatic vagotomy and intraperitoneal capsaicin desensitization on febrile phases 1–3 were assessed in adult Wistar rats. Surgical vagotomy was performed |30 d before the experiment; this procedure interrupts both afferent and efferent vagal fibers. Capsaicin was administered intraperitoneally in two consecutive injections (2 and 3 mg / kg, 3 h apart) 1 week prior to the experiment; this procedure desensitizes afferent fibers, primarily within the abdominal cavity, and does not lead to the known thermal effects of systemic capsaicin desensitization. At a neutral ambient temperature, the rats were given Escherichia coli lipopolysaccharide (10 mg / kg) through a preimplanted jugular catheter, and their colonic temperature wes measured by thermocouples for 7 h. The control rats exhibited the typical triphasic febrile responses. Confirming our earlier studies, subdiaphragmatic vagotomy did not affect phases 1 and 2; it did, however, result in a 2.5-fold reduction of phase 3. Capsaicin desensitization modified the febrile response differently: phases 2 and 3 were unaffected, but phase 1 disappeared. We suggest that neural afferent fibers (nonvagal but perhaps vagal as well) play an important role in the early febrile response (phase 1) by transducing peripheral pyrogenic signals to the brain. We also suggest that vagal efferent fibers are likely to participate in the later febrile response (phase 3) via an unknown mechanism. 2000 Elsevier Science B.V. All rights reserved. Keywords: Febrile phases; Endotoxin; Lipopolysaccharide; Vagus nerve; Subdiaphragmatic vagotomy; Capsaicin; Intra-abdominal neural afferents
The febrile response of rats to moderate-to-high doses of intravenous (i.v.) lipopolysaccharide (LPS) is polyphasic. The first three phases (phases 1, 2 and 3) of this response have been identified and are characterized by body (colonic) temperature (T c ) peaks at |1, 2, and 5 h postinjection, respectively (Romanovsky et al., 1998). The short latency of phase 1 suggests that it is mediated neurally. Afferent vagal fibers, perhaps of the hepatic branch, have been proposed as the anatomical substrate of such mediation (Maier et al., 1998; Goehler et al., 2000; Romanovsky et al., 2000b). Reportedly, truncal subdiaphragmatic vagotomy (Sehic and Blatteis, 1996; Romanovsky et al., 1997a,c), selective transection of the hepatic vagal branch (Simons et al., 1998), and capsaicin ´ desensitization of abdominal neural afferents (Szekely et *Corresponding author. Trauma Research, St. Joseph’s Hospital and Medical Center, 350 West Thomas Road, Phoenix, AZ 85013, USA. Tel.: 11-602-406-5059; fax: 11-602-406-4113. E-mail address: [email protected] (A.A. Romanovsky).
al., 1995) all attenuate the febrile response to i.v. LPS. However, the effects of vagotomy, whether surgical or ‘‘chemical’’ (capsaicin desensitization), on phases 1 and 2 of the polyphasic febrile response to i.v. or intraarterial LPS remain controversial (Sehic and Blatteis, 1996; Romanovsky et al., 1997c; Caldwell et al., 1999), whereas the effect of vagotomy on the recently discovered phase 3 has not been studied yet. The aim of the present study was to determine how febrile phases 1, 2, and 3 are affected by truncal subdiaphragmatic vagotomy (experiment 1) and capsaicin desensitization (experiment 2). The latter procedure affects only afferent C- and Ad-fibers, both vagal and nonvagal (Holzer, 1988); the former procedure interrupts both afferent and efferent vagal fibers. Importantly, truncal vagotomy does not affect most sympathetic and adventitial fibers that course with the vagus, because these fibers are abundant at the branch level but scarce in the trunks (Prechtl and Powley, 1990). Experiment 1 was conducted in 60 adult male Wistar rats (B and K Universal, Kent, WA, USA). The animals
1566-0702 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S1566-0702( 00 )00223-X
´ et al. / Autonomic Neuroscience: Basic and Clinical 85 (2000) 78 – 82 M. Szekely
were caged singly at an ambient temperature (T a ) of |228C. Food and water were available ad libitum; the vagotomized animals were given reinforced food. As in the past (Romanovsky et al., 1997a), the rats were subdiaphragmatically vagotomized (n528) or sham vagotomized (n532) |30 d before the experiment and had a catheter implanted in the right jugular vein 3 d before the experiment. The rats were extensively habituated to the experimental conditions. On the day of the test, each rat was instrumented with a colonic thermocouple connected to a data acquisitor (Model TCA-AI-24; Dianachart, Rockaway, NJ, USA) and personal computer. The animal was then placed in an individual stock and transferred to a climatic chamber (Forma Scientific, Marietta, OH, USA) set to a neutral T a of 30.08C and 50.0% relative humidity. The exteriorized portion of the i.v. catheter was passed through a wall port and connected to a syringe. After a 1-h stabilization period, measurements were begun, and T c was recorded every 2 min from 1 h before to 7 h after the i.v. injection of Escherichia coli LPS (O111:B4; Sigma, St. Louis, MO, USA) in a dose of 10 mg / kg in pyrogen-free saline (PFS; 1 ml / kg) or PFS alone. The effectiveness of surgical vagotomy was verified in six vagotomized and six sham-operated rats randomly selected from the study population. A few days after the experiment, these 12 animals were food-deprived for 24 h and thereafter euthanized with a barbiturate injection. The stomachs together with their contents were removed and weighed. Subdiaphragmatic vagotomy is known to cause gastric dilatation and inhibit the evacuatory function of the stomach, leading to increased stomach mass (Kraly et al., 1986; Romanovsky et al., 1997a). Experiment 2 was conducted in 31 adult Wistar rats ´ ´ (University Medical School Pecs, Pecs, Hungary). The animals were adapted to cold by being caged singly in standard ‘‘shoeboxes’’ with excessive woodchip bedding (to absorb moisture and allow nest building) at a T a of |58C for at least 30 d. Cold adaptation was used to increase the magnitude of the first febrile phase, the major focus of this experiment. The first phase is relatively low in nonadapted rats, but it is much higher in cold-adapted ´ rats (Szekely, unpublished data) that have been shown to develop robust thermogenic and hyperthermic responses ´ (Szekely and Mercer, 1999). One week before the experiment, the intra-abdominal neural afferents were desensitized in 14 rats by two consecutive intraperitoneal (i.p.) injections of capsaicin (8-methyl-N-vanillyl-6nonenamide; Sigma), 2 and 3 mg / kg, 3 h apart. A stock solution of capsaicin in absolute ethanol was freshly diluted with PFS to final concentrations of capsaicin and ethanol of 1 mg / ml and 10%, respectively. Low-dose i.p. desensitization was used because it causes transient (3–5 weeks) neural damage that is limited primarily to the abdominal cavity and does not lead to the known systemic ´ effects of desensitization with high doses (Szekely and Romanovsky, 1998). Sham animals (n517) received no
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capsaicin. The rats had a catheter implanted in the right jugular vein 3 d before the experiment. As in the past ´ (Szelenyi et al., 1994), each rat was instrumented with a colonic thermocouple and placed into a brass open-circuit metabolic chamber, which was immersed in a water bath. The chamber was maintained at 25.08C (a neutral T a for ´ cold-adapted rats; see Szekely and Mercer, 1999). The exteriorized portion of the i.v. catheter was passed through a wall port and connected to a syringe. After a 1-h stabilization period, the measurements were begun, and T c was recorded from 1 h before to 7 h after the i.v. injection of Escherichia coli LPS (O111:B4; 10 mg / kg) in PFS (1 ml / kg) or PFS alone. The effectiveness of capsaicin desensitization was verified in eight capsaicin-treated and 12 control rats randomly selected from the study population. A few days after the experiment, these 20 animals were food-deprived for 24 h and offered an excessive amount of standard rat chow thereafter. The animals were weighed twice, viz. immediately before and 150 min after food presentation. The difference in the body mass was calculated, expressed as a percentage of the initial body mass, and considered an index of the amount of food consumed over the 150 min. Neural (primarily vagal) afferents partially mediate satiety (Schwartz et al., 1999), and increased food consumption (satiety impairment) was expected in the capsaicin-treated animals. In both experiments, we calculated the fever index for each phase of the polyphasic febrile response as a time integral of the deviation of T c from its level at time 0 (LPS or PFS injection). The latter deviation was integrated over 30–70 min postinjection (phase 1), 70–180 min (phase 2), or 180–420 min (phase 3). The integration times were determined in advance, based on our previous data (Romanovsky et al., 1998). The fever indices were compared using Student’s unpaired t-test. This test was also used to compare stomach masses and food intakes for verification of the effectiveness of the vagotomy and capsaicin-desensitization procedures, respectively. In both experiments, all rats responded to i.v. LPS (but not PFS) with a polyphasic fever. The major results of experiment 1 are shown in Fig. 1. Compared to the sham-operated controls, the febrile response of the vagotomized rats had an identical phase 1 (7.964.2 min38C vs. 8.262.8 min38C; P55310 21 ), a similar phase 2 (60.4618.0 min38C vs. 74.1611.2 min38C; P523 10 21 ), and, on average, a |2.5-fold attenuated phase 3 (85.5653.1 min38C vs. 189.7639.5 min38C; P553 10 22 ). However, phase 3 in the vagotomized rats varied substantially: in the different runs of this experiment, it was normal, attenuated, or completely abolished compared to shams in the same subsets. The stomach mass of the vagotomized animals (3.860.4%) was |2.5-times higher than that of the shams (1.660.1%; P52310 24 ), thus confirming the effectiveness of surgery. The major results of experiment 2 are shown in Fig. 2. Compared to the controls, the capsaicin-desensitized rats
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Fig. 1. The thermal response of the subdiaphragmatically vagotomized and sham-operated rats to an intravenous injection (arrow) of lipopolysaccharide (LPS, 10 mg / kg; upper panel) or pyrogen-free saline (PFS, 1.0 ml / kg; lower panel). The initial (time 0) colonic temperatures of the vagotomized rats were 38.460.18C (LPS) and 37.960.18C (PFS). The initial colonic temperatures of the shams were 38.360.18C (LPS) and 37.960.18C (PFS).
exhibited identical phase 2 (101.8618.3 min38C vs. 102.1623.2 min38C; P55310 21 ) and phase 3 (276.9663.4 min38C vs. 276.3644.4 min38C; P553 10 21 ). However, phase 1 of their febrile response was completely abolished (5.363.8 min38C vs. 24.766.9 min38C in the controls; P58310 23 ). This febrile pattern (no or low phase 1 – normal phase 2 – normal phase 3) was consistent in all capsaicin-pretreated animals. The food consumption (increase in body mass over 150 min of postdeprivational feeding) of the capsaicin-desensitized animals (7.660.4%) was significantly higher than that of
Fig. 2. The thermal response of the capsaicin-pretreated and control cold-acclimated rats to an intravenous injection (arrow) of lipopolysaccharide (LPS, 10 mg / kg; upper panel) or pyrogen-free saline (PFS, 1.0 ml / kg; lower panel). The initial (time 0) colonic temperatures of the capsaicin-pretreated rats were 37.660.28C (LPS) and 37.760.18C (PFS). The initial colonic temperatures of the controls were 37.460.28C (LPS) and 37.560.28C (PFS).
the shams (5.460.5%; P52310 23 ), thus independently confirming the effectiveness of desensitization. Confirming our earlier speculations (Romanovsky et al., 1996), this study demonstrates that different febrile phases are mediated differently. In the present experiments, phase 1 of the polyphasic febrile response to a moderate i.v. dose of LPS was unaffected by total subdiaphragmatic vagotomy, but it was abolished by capsaicin-induced damage of intra-abdominal afferent fibers. This suggests that neural afferents are involved in the genesis of phase 1, but that nonvagal chemosensitive fibers are more important than vagal. Together with our earlier findings (Romanovsky et al., 1997a,c), the present results also indicate that phase 1 of the polyphasic febrile response to moderate doses of LPS differs from the monophasic febrile response to very low, near-threshold doses. The latter is attenuated by subdiaphragmatic vagotomy (Romanovsky et al., 1997a,c), whereas the former is unaffected (Romanovsky et al., 1997c). The relationship between monophasic fever and phase 1 of polyphasic fever is currently unclear. In the present study, phase 2 of the polyphasic LPS fever was affected neither by total truncal subdiaphragmatic vagotomy nor by i.p. capsaicin pretreatment. This observation confirms our earlier data (Romanovsky et al., 1997c) and contradicts the data of Sehic and Blatteis (1996). Although interspecies differences (rats vs. guinea pigs; see, e.g., Metz and Forssmann, 1980) may underlie this contradiction, the potential nonspecific consequences / complications of subdiaphragmatic vagotomy (Alvarez, 1948; Kraly et al., 1986), including thermoeffector insufficiency (Lin and Chern, 1985), should also be considered. In our studies, the animals were maintained on a reinforced diet after surgery; the importance of this precaution has been emphasized repeatedly (Andrews et al., 1985; Romanovsky et al., 1997a). Moreover, we have directly demonstrated good health (Romanovsky et al., 1997a) and uncompromised thermoeffector responses in these rats (Romanovsky et al., 1997b; Sugimoto et al., 1999); such controls have often been neglected by others. In sum, based on the present experiments and our earlier studies (Romanovsky et al., 1997c), we are confident that phase 2 of the polyphasic febrile response is unaffected by vagotomy, whether surgical or chemical, at least in the rat. We suggest that either intra-abdominal nerve fibers (whether afferent vagal, efferent vagal, or afferent nonvagal) are uninvolved in the genesis of phase 2 or that extra-abdominal neural and / or humoral mechanisms readily compensate for the functional inactivity of intra-abdominal fibers in vagotomized and capsaicin-desensitized animals. Recently, we have identified phase 3 of the polyphasic febrile response of rats to i.v. LPS (Romanovsky et al., 1998). The genesis of this phase remains unclear. The present study showed that phase 3 of LPS fever was unaffected by capsaicin desensitization of abdominal neural afferents but was attenuated (to various degrees) by total subdiaphragmatic vagotomy. These findings suggest that
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capsaicin-insensitive vagal fibers, presumably efferents, are involved in the genesis of the febrile phase 3, and that this involvement is strongly modulated by unknown factors that were uncontrolled in the present experiments. Interestingly, the involvement of efferent vagal fibers in i.p. LPS-induced fever (Romanovsky et al., 2000a) and i.v. LPS-induced shock (Borovikova et al., 2000) has recently been demonstrated. We conclude that the three phases of the polyphasic febrile response of rats to i.v. LPS are differentially sensitive to vagotomy and, therefore, likely to be mediated differently. Phase 1 is abolished by capsaicin desensitization of intra-abdominal neural afferents but unaffected by truncal subdiaphragmatic vagotomy. Phase 2 is unchanged in both surgically vagotomized and capsaicin-desensitized rats. Phase 3 is unaffected by i.p. capsaicin pretreatment but attenuated by subdiaphragmatic vagotomy. We speculate that some intra-abdominal neural afferents are involved in the genesis of phase 1, and that nonvagal afferent fibers are more important for this phase than vagal afferents. We further suggest that intra-abdominal nerve fibers are uninvolved in the genesis of phase 2 or that other mechanisms readily compensate for their functional inactivity in vagotomized and capsaicin-desensitized animals. Finally, we propose that vagal efferents (but not vagal or nonvagal afferents) are involved in the genesis of the febrile phase 3, and that this involvement is strongly modulated by as of yet unidentified factors. The question ‘‘Is the vagus nerve involved in the febrile response?’’ is probably too general to be answered correctly. In the case of i.v. LPS-induced fever in rats, this question should be rephrased as follows: ‘‘Which type of neural fibers (afferent or efferent; vagal or nonvagal) is involved in each febrile phase?’’ Together with other papers in this issue, the present results suggest that multiple neural mechanisms underlie the fever response.
Acknowledgements The authors thank Dr. S. Kick and Ms. D. Mutchler for editing the manuscript. This study was supported in part by grants and donations from the National Institutes of Health (1 RO1 NS41233-01), Oregon Health Sciences Foundation (Medical Research Foundation of Oregon), Collins Medical Trust, Good Samaritan Foundation, Bayer AG, and Dr. Temple Fay Memorial Account to A.A.R. and from the Hungarian Scientific Research Grant Agency (OTKA T026511) to M.S. V.A.K. was on leave from the Institute of Physiology, Minsk, Belarus.
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