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Fasting is a physiological stimulus of vagus-mediated enhancement of nociception in the female rat
Neuroscience 119 (2003) 215–221
FASTING IS A PHYSIOLOGICAL STIMULUS OF VAGUS-MEDIATED ENHANCEMENT OF NOCICEPTION IN THE FEMALE RAT S. G. KHASAR, D. B. REICHLING, P. G. GREEN, W. M. ISENBERG AND J. D. LEVINE*
1994; Ja¨nig et al., 2000). In a previous study, we found that sectioning the subdiaphragmatic branch of the vagus reduces nociception in the formalin test in male rats, and this effect of vagotomy depends on the adrenal medulla and gonadal hormones (Khasar et al., 2001). Our initial characterization of this vagal/gonadal system relied on nonphysiological (surgical) manipulations. However, we speculated that physiological changes in the level of activity in the subdiaphragmatic vagus might be sufficient to dynamically regulate nociception via this pathway. Because the subdiaphragmatic branch of the vagus, in particular, is crucial for the phenomenon, we suggested that activity originating from the gut might be most effective. In the present study we used short-term food deprivation (fasting) as a natural stimulus to increase activity in the subdiaphragmatic vagus. Our previous observations on the effect of vagotomy (Khasar et al., 2001) indicated that the vagus-dependent pain modulation pathway may be tonically active in male rats, while in female rats the baseline level of activity may be low. Therefore, the present experiments, to determine if the pathway can be activated by fasting, focus on the effect in female rats.
Departments of Oral and Maxillofacial Surgery, Medicine, and Anatomy, and Program in Neuroscience, Room C-522, Campus Box 0440, NIH Pain Center, University of California, San Francisco CA 941430440, USA
Abstract—The vagus nerve modulates nociception by a mechanism dependent upon gonadal hormones and the adrenal medulla. In the present study we tested the hypothesis that this modulation is dynamically controlled by physiological stimulation of structures innervated by the subdiaphragmatic vagus. Specifically, food deprivation (fasting) was employed to increase activity in the subdiaphragmatic vagus, and the experiments were performed mainly in female rats because our previous observations suggested that baseline activity in the pathway is lower in females than in males. Consistent with the hypothesis, after a 48-h fast, female rats exhibited increased nociceptive behavior in the formalin test. In contrast, fasting had no effect on formalin-evoked nociceptive behavior in male rats. The fasting-induced effect on nociception appears to be mediated by the vagus nerve since it is prevented by subdiaphragmatic vagotomy. Also similar to the previously characterized vagus-mediated modulation, the effect of fasting in the female is blocked by gonadectomy or adrenal medullectomy, and hormone replacement with 17-estradiol in gonadectomized female rats restored the effect of fasting. Decreased glucose metabolism apparently does not play a significant role in the effect of fasting on nociception, since the effect was unchanged when 5% glucose was provided in the drinking water throughout the fasting period. On the other hand, increasing the bulk content of the stomach (without providing nutrients) by infusion of petrolatum significantly attenuated the effect of fasting during the interphase period of the formalin response, suggesting that decreased gut distention, and possibly motility, are important in fasting-induced enhancement of nociception. These results indicate that fasting is a physiological activator of the vagus-mediated pain modulation pathway. This suggests the possibility that, especially in females, natural periodic changes in gut distention and motility may control an ongoing vagus-mediated adjustment in the organism’s nociceptive sensitivity. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
EXPERIMENTAL PROCEDURES Experiments were performed on age-matched (6 – 8 weeks old) male (140 –250 g) and female (100 –170 g) Sprague–Dawley rats (Bantin and Kingman, Fremont, CA, USA) housed in the Laboratory Animal Resource Center of the University of California, San Francisco, under a 12-h light/dark cycle. Care and use of rats conformed to National Institutes of Health guidelines and experimental protocols were approved by the University of California, San Francisco Committee on Animal Research. In order to minimize animal suffering, a minimum number was used. Also anesthesia was used during all surgical operations.
Fasting Food was withdrawn 48 h prior to performing the formalin test, but water continued to be available ad libitum. Control rats had free access to both food and water. Plasma glucose levels were measured in blood from the tail vein using a One-Touch Basic meter (LifeScan, Milpitas, CA, USA).
Key words: chronic pain, hyperalgesia, parasympathetic nervous system, estrogen, sex, gender.
Formalin test One hour prior to performing the formalin test, rats were habituated to the acrylic box (78.7 cm⫻78.7 cm⫻81.3 cm) in which nociceptive behaviors were observed. Formalin (50 l; 1% in physiological saline) was injected into the plantar surface of the left hind paw. Behavioral scoring was performed by a modification of the method of Dubuisson and Dennis, which employed six behavioral categories rather than the original four (Dubuisson and Dennis, 1977). Thus, behavior was scored as follows: 0⫽injected paw flat on the floor; 1⫽side of injected paw on the floor; 2⫽heel
Nociception is modulated by the vagus nerve (Gebhart and Randich, 1992; Randich and Gebhart, 1992; Watkins et al., *Corresponding author. Tel: ⫹1-415-476-5108; fax: ⫹1-415-4766305. E-mail address: [email protected] (J. D. Levine). Abbreviations: NTS, nucleus of the solitary tract; PVN, paraventricular nucleus.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00136-2
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of injected paw on the floor; 3⫽injected paw elevated; 4⫽injected paw shaken; and 5⫽injected paw licked or bitten. Scores were manually keyed into a computer for the duration of the 60-min observation period. The nociceptive behavioral score for each 3-min interval was calculated as the weighted average of the number of seconds spent in each behavior.
Intragastric administration of petrolatum To increase bulk contents of the stomach without providing nutrients, petrolatum was administered by gastric gavage. A 5-ml volume of petrolatum (Weigert et al., 1997) was infused at a rate of approximately 2 ml/min in awake rats. Similarly, sham gavage was performed in a control group, with no pressure applied to the feeding tube. Infusions were performed at 10, 24, 34 and 46 h following the initiation of fasting. and the formalin test was then performed 2 h after the final petrolatum infusion or after sham gavage.
Surgery Surgical removal of a segment of the vagus nerve below the diaphragm was accomplished as previously described (Prechtl and Powley, 1985). In brief, a ventral incision was made in the lateral abdominal wall. The subdiaphragmatic vagus nerve was dissected free from the esophagus and blood vessels and a 1.5–2.0-cm segment of the nerve, together with its fine branches, was removed. Adrenal medullae were removed from isoflurane-anesthetized rats via an incision in the lateral abdominal wall. The adrenal capsule was incised and the adrenal medulla removed (Wilkinson et al., 1981; Miao et al., 1992). Rats were provided with 0.5% saline to drink, in place of water, for the first 7 days after surgery. This surgery was performed at least 5 weeks before formalin testing to allow the function of hypothalamo–pituitary–adrenal axis to recover (Wilkinson et al., 1981). Ovariectomy was performed on 21-day-old female rats (before onset of puberty) by methods described previously (Waynforth and Flecknell, 1992). Briefly, under ether anesthesia, an incision was made on the dorsal midline followed by bilateral incisions through the peritoneum. The vascular bundles of both ovaries were ligated, and the ovaries were excised. The animals were used for fasting experiments 3 weeks after surgery.
Administration of gonadal steroids Chronic administration of female gonadal steroids was achieved using implants that have been shown to produce sustained physiological levels of gonadal steroids (Bridges, 1984). As described previously (Smith et al., 1977; Green et al., 1999), implants were fabricated using Silastic tubing capped with Silastic plugs (1.67-mm inner diameter and 3.18-mm outer diameter; Goodfellow, Berwyn, PA, USA). Estrogen implants consisted of 5-mm segments filled with 17-estradiol. Implants were washed in absolute ethanol and equilibrated in four changes of warm phosphate-buffered saline over a 24-h period before implantation in the rat. At the time of gonadectomy, under isoflurane anesthesia (1.5–2.0% in oxygen), implants were placed under the skin of the back. In some rats, vagotomy was performed 2 weeks following gonadectomy and/or implant placement.
Materials The following chemicals were used: formaldehyde (37% w/w; Fisher Scientific, South San Francisco, CA, USA); d-glucose and 17-estradiol (1,3,5[10]-estratriene-3, 17-diol; Sigma Chemical Co., St. Louis, MO, USA). Formalin solution was made by diluting formaldehyde in normal saline.
Fig. 1. Fasting enhances the nociceptive response to formalin in female rats. (A) Following a 48-h period of fasting, female rats exhibited an increased occurrence of nociceptive behaviors in the formalin test during phase 1, the interphase and phase 2. (B) In contrast, in male rats there was a small but significant increase only during phase 2.
Statistical analyses Data are presented as mean⫾S.E.M. and analyzed by one-way analysis of variance. Phase 1 (0 –3 min), the interphase (9 –15 min) and phase 2 (18 – 60 min) of the formalin test were analyzed separately, using one time point of each phase: 3 min for phase 1, 12 min for the interphase, and 60 min for phase 2. Scheffe´’s post hoc test was employed to determine where significant differences occurred. The defined level for statistical significance was P⬍0.05.
RESULTS Pro-nociceptive effect of fasting Fasted female rats were compared with non-fasted controls to determine if the fasted rats were significantly more responsive to formalin. As shown in Fig. 1A, normal female rats responded to injection of 1% formalin into the left plantar hind paw with a typical two-phase period of nociceptive behaviors (Dubuisson and Dennis, 1977).
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Following a 48-h fast, female rats exhibited enhanced nociceptive behavior in response to formalin. Specifically, there were significant differences in behavioral response between fasted (n⫽10) and non-fasted (n⫽6) control rats during phase 1 (F(1,14)⫽6.308; P⫽0.025), the interphase (F(1,14)⫽107.837; P⬍0.001) and phase 2 (F(1,14)⫽72.146; P⬍0.001). In contrast, in male rats there was a small, albeit significant, difference in formalin-induced nociceptive behavior between fasted (n⫽6) and non-fasted (n⫽6) controls only during phase 2 (P⫽0.040; Fig. 1B). Physiologically relevant consequences of fasting To determine if the effect of fasting is reversible, one group of female rats (n⫽6) was fasted for 48 h, after which the group was allowed access to food ad libitum for a period of 48 h. At the end of the feeding period the formalin test was performed. As shown in Fig. 2A, there was no significant difference between rescue-fed rats and control non-fasted rats during phase 1 (F(1,10)⫽0.301; P⫽0.595), the interphase (F(1,10)⫽2,242; P⫽0.165) or phase 2 (F(1,10)⫽1.123; P⫽0.314). This result suggests that fasting-induced hyperalgesia in female rats is not due to any long-term change in the condition of the animal that might be caused by reduced food intake. Further experiments were performed to investigate which physiological effects of fasting are most relevant to the observed effect on nociception. Hypoglycemia is an important consequence of acute fasting (Bequet et al., 2000); the fasting protocol in this study caused a decrease in plasma glucose levels from 90.7⫾1.6 mg/dl before fasting (n⫽18) to 43.6⫾2 mg/dl at the end of the 48-h fasting period (n⫽12). Therefore, to determine if reduced glucose metabolism contributes to the fasting-induced enhancement of nociception, supplemental glucose was provided throughout the 48-h fasting period (5% glucose in the drinking water ad libitum) to one group of rats (n⫽6). At the end of the 48-h fasting period plasma glucose levels in glucose-supplemented rats (77⫾4 mg/dl; n⫽6) were close to the pre-fasting level. As shown in Fig. 2B, glucose supplementation caused no significant change in the fasting-enhanced response to formalin during phase 1 (F(1,14)⫽0.011; P⫽0.920), the interphase (F(1,14)⫽0.708; P⫽0.414) or phase 2 (F(1,14)⫽0.047; P⫽0.832). This suggests that altered glucose metabolism does not play an essential role in the observed fasting-induced enhancement of nociception. Another physiological consequence of fasting is altered gastrointestinal motility due to the absence of bulk contents in the gut. To test if this phenomenon contributes to the nociception-related effect of fasting, intragastric infusion of a volume of petrolatum was used to produce stomach distention (Weigert et al., 1997) without changing the nutritional status of the animal. One group of fasted rats (n⫽7) received four infusions of petrolatum during the fasting period. As shown in Fig. 2C, compared with the sham-fed control group (n⫽8), this procedure significantly reduced the fasting-induced enhancement of formalin nociceptive behavior during the interphase (F(1,13)⫽4.883; P⫽0.046) but there was not
Fig. 2. The effect of fasting may depend on changes in gut distension and motility, but not on altered glucose metabolism. (A) Female rats that had been fasted for 48 h then provided with food for 48 h did not exhibit any significant enhancement of nociceptive behaviors in the formalin test. (B) Supplementation of the drinking water with 5% glucose during the fasting period did not change the effect of fasting in female rats. (C) Intragastric administration of non-nutritional bulk (5 ml petrolatum) significantly reduced the pronociceptive effect of fasting in female rats during phase 1 and the interphase.
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a significant change during phase 1 (F(1,13)⫽2.8; P⫽0.117) or phase 2 (F(1,13)⫽0.346; P⫽0.566). Role of vagus-dependent mechanisms The hypothesis that fasting would enhance nociception was based on our previous characterization of a vagusdependent pain-modulation pathway that involves adrenal medulla and gonadal hormones. Therefore, experiments were performed to test if indeed that vagal pathway underlies the effect of fasting. To test if fasting-induced enhancement of nociception requires the vagus nerve, in one group of rats (n⫽6) the subdiaphragmatic branch of the vagus was sectioned two weeks before the beginning of the 48-h fasting period. As shown in Fig. 3A, compared with control (sham surgery) fasted rats (n⫽6), nociceptive response to formalin in vagotomized fasted rats was significantly reduced during the interphase (F(2,15)⫽16.369; P⬍0.001), but not during phase 1 or phase 2. Next, to determine if fasting-induced enhancement of nociception requires the adrenal medulla, adrenal medullectomy was performed in one group of rats (n⫽6). As shown in Fig. 3A, compared with control (sham surgery)fasted rats, the nociceptive response to formalin in adrenal medullectomized fasted rats was significantly reduced during the interphase (n⫽6; P⫽0.132), but not during phase 1 or phase 2. Finally, we investigated the role of gonadal hormones in fasting-induced enhancement of nociception. As shown in Fig. 3B, orchidectomy prevented the fasting-induced enhancement of nociceptive response to formalin, so that there was no significant difference between the response to formalin in fasted (n⫽8) versus non-fasted (n⫽8) female rats. Administration of 17-estradiol to orchidectomized rats (n⫽8), reconstituted the fasting-induced enhancement of the behavioral response to formalin. There were significant differences between the groups during all phases of the formalin test: phase 1 (F(2,21)⫽4.094; P⫽0.032), interphase (F(2,21)⫽27.223; P⬍0.001) and phase 2 (F(2,21)⫽6.103; P⫽0.008). Post hoc tests showed that during phase 1, the orchidectomized, estradiol-treated fasted group was significantly different only from the orchidectomized non-fasted group (P⫽0.04), but during the interphase, the orchidectomized, estradiol-treated fasted group was significantly different from both orchidectomized groups (P⬍0.001 each). Also during phase 2, the orchidectomized, estradiol-treated fasted group was significantly different from the orchidectomized non-fasted (P⫽0.037) and the orchidectomized fasted group (P⫽0.015) groups.
DISCUSSION This study demonstrates that, in female rats, fasting can act as a physiological stimulus of the recently described endogenous nociceptive modulation pathway involving the vagus, adrenal medulla, and gonadal hormones (Khasar et al., 2001). For most organisms existing in the wild, brief periods of fasting are not unusual. This suggests that the
Fig. 3. The effect of fasting in female rats involves the vagus nerve, adrenal medulla and gonadal hormones. (A) Sectioning the vagus nerve significantly reduced fasting-enhanced behavioral responses in the formalin test during the interphase interval. Removal of the adrenal medullae also attenuated the fasting-induced enhancement of responses in the formalin test. Sham abdominal surgery had no significant effect on the effect of fasting. (B) Removal of the ovaries also prevented the fasting-induced enhancement of responses in the formalin test during the interphase. Thus, there were no significant differences in the responses between orchidectomized rats that were fasted and not fasted. The effect of orchidectomy was reversed by administration of 17-estradiol.
pathway functions not merely as a static control over pain sensitivity, but that it is adapted to dynamically modulate nociception. The pro-nociceptive effect of fasting was much greater in female than male rats. This sex-dependence is consistent with the idea that the vagus-dependent nociceptive modulation pathway is tonically active in males but much less active in females (based on the observation that vagotomy increases pain threshold in males but has little effect in females; Khasar et al., 2001). It is possible that under more naturalistic conditions where food is not available at all times ad libitum, females might also exhibit some baseline activation of the system. Under such con-
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ditions, the system might be capable of performing ongoing up- and down-adjustments in nociceptive sensitivity in response to the continually changing feeding status and hormonal levels of the animal. In this regard it would be of interest for future studies to examine the time course and stimulus intensity-response relationship for fasting-induced modulation of nociception. The present data indicate that increases and decreases in vagus-dependent pain enhancement can take place in less than 48 h. Because vagotomy reversed the effect of fasting on nociception, we suggest that enhancement of nociception by fasting may be caused by an increase in vagal afferent activity. Consistent with this idea, it has been reported that vagal activity increases during fasting (Szekely et al., 2000). There is evidence that activity in different vagal afferents can be either pro- or anti-nociceptive (Gebhart and Randich, 1992; Randich and Gebhart, 1992; Watkins et al., 1994). We hypothesize that fasting activates vagal afferents that are pro-nociceptive with respect to formalininduced behaviors. This contrasts with the effect of vagal afferent stimulation on bradykinin-induced hyperalgesia, which we showed previously is anti-nociceptive (Khasar et al., 1998a,b). Although some vagal afferents are glucoreceptors (Paintal, 1973; Mei, 1985), it seems unlikely that these contribute to the effects observed in the present experiments, since hypoglycemia during fasting (Bequet et al., 2000) would tend to decrease activity in these fibers. Previous studies using normal, non-fasted, male rats have reported effects of sucrose ingestion on nociception (Roane and Martin, 1990; Frye et al., 1993; d’Anci et al., 1996; Kanarek et al., 1997, 2001; Mukherjee et al., 2000; Mukherjee et al., 2002), including analgesia in formalininduced nociceptive behaviors (Mukherjee et al., 2000; Dutta et al., 2001). However, our finding that glucose supplementation does not prevent the fasting-induced enhancement of nociception suggests that altered glucose metabolism does not play an essential role in the effect. On the other hand, we found that petrolatum gavage reversed fasting-induced enhancement of nociception (during the interphase interval of the formalin response) while sham gavage had no effect. This reversal might be explained by a gavage-induced decrease of activity in mechanoreceptive vagal afferents (Andrews et al., 1980; Blackshaw et al., 1987; Davison and Clarke, 1988). Specifically, the petrolatum gavage might cause a decrease in activity by triggering the decrease in fasting-induced cyclic motor activity such as typically occurs in the duodenum upon end-of-fast feeding (Kihara et al., 2001). Studies by Watkins and coworkers (Watkins et al., 1994) suggested that vagal activity may play an important role in producing the systemic hyperalgesia associated with illness. However, those investigators found no effect of vagotomy on formalin-induced nociception. This apparent contradiction with our observations probably reflects the use of male rats only by Watkins et al. The interplay between vagal activity, fasting, and nociception in a natural state of sickness may be quite complex, with vagus-dependent hyperalgesia and illness promoting anorexia
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(Konsman and Dantzer, 2001). Our results suggest that anorexia-induced fasting could, in turn, further exacerbate the systemic hyperalgesia associated with illness. Subdiaphragmatic vagal afferents converge on the nucleus of the solitary tract (NTS), which has projections to the paraventricular nuleus (PVN) (Berthoud and Neuhuber, 2000), and the adrenal medulla is innervated by preganglionic sympathetic nerves that receive input from the PVN (Motawei et al., 1999). The PVN plays a very important role in the regulation of feeding, satiety and fasting (Leibowitz, 1988; Maeda et al., 1996) as well as nociception (Snowball et al., 2000; Hallbeck, 2000; Palkovits et al., 1999). We hypothesize that fasting stimulates vagal afferent activity that is transmitted to the NTS, from which it is relayed to the PVN and the adrenal medulla. We have previously shown that vagal modulation of tonic nociception is sexually dimorphic (Khasar et al., 2001), and that adrenal medulla-dependent regulation of the inflammatory response is sexually dimorphic (Green et al., 1999, 2001). Maeda and colleagues have detected estrogen receptors in the NTS and in the PVN, that are up-regulated during fasting (Maeda et al., 1996) and there is evidence that plasma estrogen levels also increase during fasting (Otukonyong et al., 2000). Thus, with presence of estrogen receptors on nodose ganglion (vagal afferent) neurons (Papka et al., 1997, 2001), the NTS, the PVN (Maeda et al., 1996) and the adrenal medulla (Green et al., 1999), the neural pathway proposed to mediate fastinginduced enhancement of formalin-induced nociceptive behaviors is sexually dimorphic at several levels. Although petrolatum gavage prevented the effect of fasting during the interphase, it had no significant effect on the fasting-enhanced behavior during phase 1 or phase 2. It is thought that phase 1 may be caused by increased activity in primary afferent nociceptors due to their direct activation by formalin (Hunskaar and Hole, 1987) as well as by inflammatory mediator-induced nociceptor sensitization (Correa and Calixto, 1993; Doak and Sawynok, 1997; Parada et al., 2001). Phase 2, in addition to increased activity of sensitized nociceptors, may also involve sensitization of nociceptive circuitry in the CNS (Dickenson and Sullivan, 1987; Coderre and Melzack, 1992). The decrease in nociceptive behavior that ordinarily occurs during the interphase, the period between phases 1 and 2, is thought to reflect active inhibition of nociceptive transmission in the spinal cord (Franklin and Abbott, 1993; Kaneko and Hammond, 1997; Omote et al., 1998; Henry et al., 1999). Therefore, we speculate that the decrease in fasting-induced behavior during the interphase interval, caused by petrolatum infusion, may involve a restoration of spinal inhibitory mechanisms that would normally operate during that period. As the circuitry underlying interphase inhibition becomes better understood, it will be of great interest to investigate possible sexual dimorphism in those pathways, as well as interactions with vagal and adrenal mechanisms implicated in the effect of fasting. In summary, we have shown that fasting can act as a physiological stimulus of the endogenous pain-modulation pathway involving the vagus, adrenal medulla, and go-
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nadal hormones. This system may be active during periods of fasting, whether caused by food shortage or perhaps motivated by illness. Furthermore, we can speculate that the system might become particularly important in adjusting pain sensitivity in female rats during the process of reproduction, when gonadal hormones, vagal activity, and nociception are altered. Acknowledgements—The authors are grateful to Drs. Steven Paul and Robert Gear for assistance with statistical analyses, and to Mr. Dennis Mendoza for technical assistance. This research was supported by the National Institutes of Health grant NR04880.
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(Accepted 31 December 2002)
FASTING IS A PHYSIOLOGICAL STIMULUS OF VAGUS-MEDIATED ENHANCEMENT OF NOCICEPTION IN THE FEMALE RAT S. G. KHASAR, D. B. REICHLING, P. G. GREEN, W. M. ISENBERG AND J. D. LEVINE*
1994; Ja¨nig et al., 2000). In a previous study, we found that sectioning the subdiaphragmatic branch of the vagus reduces nociception in the formalin test in male rats, and this effect of vagotomy depends on the adrenal medulla and gonadal hormones (Khasar et al., 2001). Our initial characterization of this vagal/gonadal system relied on nonphysiological (surgical) manipulations. However, we speculated that physiological changes in the level of activity in the subdiaphragmatic vagus might be sufficient to dynamically regulate nociception via this pathway. Because the subdiaphragmatic branch of the vagus, in particular, is crucial for the phenomenon, we suggested that activity originating from the gut might be most effective. In the present study we used short-term food deprivation (fasting) as a natural stimulus to increase activity in the subdiaphragmatic vagus. Our previous observations on the effect of vagotomy (Khasar et al., 2001) indicated that the vagus-dependent pain modulation pathway may be tonically active in male rats, while in female rats the baseline level of activity may be low. Therefore, the present experiments, to determine if the pathway can be activated by fasting, focus on the effect in female rats.
Departments of Oral and Maxillofacial Surgery, Medicine, and Anatomy, and Program in Neuroscience, Room C-522, Campus Box 0440, NIH Pain Center, University of California, San Francisco CA 941430440, USA
Abstract—The vagus nerve modulates nociception by a mechanism dependent upon gonadal hormones and the adrenal medulla. In the present study we tested the hypothesis that this modulation is dynamically controlled by physiological stimulation of structures innervated by the subdiaphragmatic vagus. Specifically, food deprivation (fasting) was employed to increase activity in the subdiaphragmatic vagus, and the experiments were performed mainly in female rats because our previous observations suggested that baseline activity in the pathway is lower in females than in males. Consistent with the hypothesis, after a 48-h fast, female rats exhibited increased nociceptive behavior in the formalin test. In contrast, fasting had no effect on formalin-evoked nociceptive behavior in male rats. The fasting-induced effect on nociception appears to be mediated by the vagus nerve since it is prevented by subdiaphragmatic vagotomy. Also similar to the previously characterized vagus-mediated modulation, the effect of fasting in the female is blocked by gonadectomy or adrenal medullectomy, and hormone replacement with 17-estradiol in gonadectomized female rats restored the effect of fasting. Decreased glucose metabolism apparently does not play a significant role in the effect of fasting on nociception, since the effect was unchanged when 5% glucose was provided in the drinking water throughout the fasting period. On the other hand, increasing the bulk content of the stomach (without providing nutrients) by infusion of petrolatum significantly attenuated the effect of fasting during the interphase period of the formalin response, suggesting that decreased gut distention, and possibly motility, are important in fasting-induced enhancement of nociception. These results indicate that fasting is a physiological activator of the vagus-mediated pain modulation pathway. This suggests the possibility that, especially in females, natural periodic changes in gut distention and motility may control an ongoing vagus-mediated adjustment in the organism’s nociceptive sensitivity. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
EXPERIMENTAL PROCEDURES Experiments were performed on age-matched (6 – 8 weeks old) male (140 –250 g) and female (100 –170 g) Sprague–Dawley rats (Bantin and Kingman, Fremont, CA, USA) housed in the Laboratory Animal Resource Center of the University of California, San Francisco, under a 12-h light/dark cycle. Care and use of rats conformed to National Institutes of Health guidelines and experimental protocols were approved by the University of California, San Francisco Committee on Animal Research. In order to minimize animal suffering, a minimum number was used. Also anesthesia was used during all surgical operations.
Fasting Food was withdrawn 48 h prior to performing the formalin test, but water continued to be available ad libitum. Control rats had free access to both food and water. Plasma glucose levels were measured in blood from the tail vein using a One-Touch Basic meter (LifeScan, Milpitas, CA, USA).
Key words: chronic pain, hyperalgesia, parasympathetic nervous system, estrogen, sex, gender.
Formalin test One hour prior to performing the formalin test, rats were habituated to the acrylic box (78.7 cm⫻78.7 cm⫻81.3 cm) in which nociceptive behaviors were observed. Formalin (50 l; 1% in physiological saline) was injected into the plantar surface of the left hind paw. Behavioral scoring was performed by a modification of the method of Dubuisson and Dennis, which employed six behavioral categories rather than the original four (Dubuisson and Dennis, 1977). Thus, behavior was scored as follows: 0⫽injected paw flat on the floor; 1⫽side of injected paw on the floor; 2⫽heel
Nociception is modulated by the vagus nerve (Gebhart and Randich, 1992; Randich and Gebhart, 1992; Watkins et al., *Corresponding author. Tel: ⫹1-415-476-5108; fax: ⫹1-415-4766305. E-mail address: [email protected] (J. D. Levine). Abbreviations: NTS, nucleus of the solitary tract; PVN, paraventricular nucleus.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00136-2
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of injected paw on the floor; 3⫽injected paw elevated; 4⫽injected paw shaken; and 5⫽injected paw licked or bitten. Scores were manually keyed into a computer for the duration of the 60-min observation period. The nociceptive behavioral score for each 3-min interval was calculated as the weighted average of the number of seconds spent in each behavior.
Intragastric administration of petrolatum To increase bulk contents of the stomach without providing nutrients, petrolatum was administered by gastric gavage. A 5-ml volume of petrolatum (Weigert et al., 1997) was infused at a rate of approximately 2 ml/min in awake rats. Similarly, sham gavage was performed in a control group, with no pressure applied to the feeding tube. Infusions were performed at 10, 24, 34 and 46 h following the initiation of fasting. and the formalin test was then performed 2 h after the final petrolatum infusion or after sham gavage.
Surgery Surgical removal of a segment of the vagus nerve below the diaphragm was accomplished as previously described (Prechtl and Powley, 1985). In brief, a ventral incision was made in the lateral abdominal wall. The subdiaphragmatic vagus nerve was dissected free from the esophagus and blood vessels and a 1.5–2.0-cm segment of the nerve, together with its fine branches, was removed. Adrenal medullae were removed from isoflurane-anesthetized rats via an incision in the lateral abdominal wall. The adrenal capsule was incised and the adrenal medulla removed (Wilkinson et al., 1981; Miao et al., 1992). Rats were provided with 0.5% saline to drink, in place of water, for the first 7 days after surgery. This surgery was performed at least 5 weeks before formalin testing to allow the function of hypothalamo–pituitary–adrenal axis to recover (Wilkinson et al., 1981). Ovariectomy was performed on 21-day-old female rats (before onset of puberty) by methods described previously (Waynforth and Flecknell, 1992). Briefly, under ether anesthesia, an incision was made on the dorsal midline followed by bilateral incisions through the peritoneum. The vascular bundles of both ovaries were ligated, and the ovaries were excised. The animals were used for fasting experiments 3 weeks after surgery.
Administration of gonadal steroids Chronic administration of female gonadal steroids was achieved using implants that have been shown to produce sustained physiological levels of gonadal steroids (Bridges, 1984). As described previously (Smith et al., 1977; Green et al., 1999), implants were fabricated using Silastic tubing capped with Silastic plugs (1.67-mm inner diameter and 3.18-mm outer diameter; Goodfellow, Berwyn, PA, USA). Estrogen implants consisted of 5-mm segments filled with 17-estradiol. Implants were washed in absolute ethanol and equilibrated in four changes of warm phosphate-buffered saline over a 24-h period before implantation in the rat. At the time of gonadectomy, under isoflurane anesthesia (1.5–2.0% in oxygen), implants were placed under the skin of the back. In some rats, vagotomy was performed 2 weeks following gonadectomy and/or implant placement.
Materials The following chemicals were used: formaldehyde (37% w/w; Fisher Scientific, South San Francisco, CA, USA); d-glucose and 17-estradiol (1,3,5[10]-estratriene-3, 17-diol; Sigma Chemical Co., St. Louis, MO, USA). Formalin solution was made by diluting formaldehyde in normal saline.
Fig. 1. Fasting enhances the nociceptive response to formalin in female rats. (A) Following a 48-h period of fasting, female rats exhibited an increased occurrence of nociceptive behaviors in the formalin test during phase 1, the interphase and phase 2. (B) In contrast, in male rats there was a small but significant increase only during phase 2.
Statistical analyses Data are presented as mean⫾S.E.M. and analyzed by one-way analysis of variance. Phase 1 (0 –3 min), the interphase (9 –15 min) and phase 2 (18 – 60 min) of the formalin test were analyzed separately, using one time point of each phase: 3 min for phase 1, 12 min for the interphase, and 60 min for phase 2. Scheffe´’s post hoc test was employed to determine where significant differences occurred. The defined level for statistical significance was P⬍0.05.
RESULTS Pro-nociceptive effect of fasting Fasted female rats were compared with non-fasted controls to determine if the fasted rats were significantly more responsive to formalin. As shown in Fig. 1A, normal female rats responded to injection of 1% formalin into the left plantar hind paw with a typical two-phase period of nociceptive behaviors (Dubuisson and Dennis, 1977).
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Following a 48-h fast, female rats exhibited enhanced nociceptive behavior in response to formalin. Specifically, there were significant differences in behavioral response between fasted (n⫽10) and non-fasted (n⫽6) control rats during phase 1 (F(1,14)⫽6.308; P⫽0.025), the interphase (F(1,14)⫽107.837; P⬍0.001) and phase 2 (F(1,14)⫽72.146; P⬍0.001). In contrast, in male rats there was a small, albeit significant, difference in formalin-induced nociceptive behavior between fasted (n⫽6) and non-fasted (n⫽6) controls only during phase 2 (P⫽0.040; Fig. 1B). Physiologically relevant consequences of fasting To determine if the effect of fasting is reversible, one group of female rats (n⫽6) was fasted for 48 h, after which the group was allowed access to food ad libitum for a period of 48 h. At the end of the feeding period the formalin test was performed. As shown in Fig. 2A, there was no significant difference between rescue-fed rats and control non-fasted rats during phase 1 (F(1,10)⫽0.301; P⫽0.595), the interphase (F(1,10)⫽2,242; P⫽0.165) or phase 2 (F(1,10)⫽1.123; P⫽0.314). This result suggests that fasting-induced hyperalgesia in female rats is not due to any long-term change in the condition of the animal that might be caused by reduced food intake. Further experiments were performed to investigate which physiological effects of fasting are most relevant to the observed effect on nociception. Hypoglycemia is an important consequence of acute fasting (Bequet et al., 2000); the fasting protocol in this study caused a decrease in plasma glucose levels from 90.7⫾1.6 mg/dl before fasting (n⫽18) to 43.6⫾2 mg/dl at the end of the 48-h fasting period (n⫽12). Therefore, to determine if reduced glucose metabolism contributes to the fasting-induced enhancement of nociception, supplemental glucose was provided throughout the 48-h fasting period (5% glucose in the drinking water ad libitum) to one group of rats (n⫽6). At the end of the 48-h fasting period plasma glucose levels in glucose-supplemented rats (77⫾4 mg/dl; n⫽6) were close to the pre-fasting level. As shown in Fig. 2B, glucose supplementation caused no significant change in the fasting-enhanced response to formalin during phase 1 (F(1,14)⫽0.011; P⫽0.920), the interphase (F(1,14)⫽0.708; P⫽0.414) or phase 2 (F(1,14)⫽0.047; P⫽0.832). This suggests that altered glucose metabolism does not play an essential role in the observed fasting-induced enhancement of nociception. Another physiological consequence of fasting is altered gastrointestinal motility due to the absence of bulk contents in the gut. To test if this phenomenon contributes to the nociception-related effect of fasting, intragastric infusion of a volume of petrolatum was used to produce stomach distention (Weigert et al., 1997) without changing the nutritional status of the animal. One group of fasted rats (n⫽7) received four infusions of petrolatum during the fasting period. As shown in Fig. 2C, compared with the sham-fed control group (n⫽8), this procedure significantly reduced the fasting-induced enhancement of formalin nociceptive behavior during the interphase (F(1,13)⫽4.883; P⫽0.046) but there was not
Fig. 2. The effect of fasting may depend on changes in gut distension and motility, but not on altered glucose metabolism. (A) Female rats that had been fasted for 48 h then provided with food for 48 h did not exhibit any significant enhancement of nociceptive behaviors in the formalin test. (B) Supplementation of the drinking water with 5% glucose during the fasting period did not change the effect of fasting in female rats. (C) Intragastric administration of non-nutritional bulk (5 ml petrolatum) significantly reduced the pronociceptive effect of fasting in female rats during phase 1 and the interphase.
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a significant change during phase 1 (F(1,13)⫽2.8; P⫽0.117) or phase 2 (F(1,13)⫽0.346; P⫽0.566). Role of vagus-dependent mechanisms The hypothesis that fasting would enhance nociception was based on our previous characterization of a vagusdependent pain-modulation pathway that involves adrenal medulla and gonadal hormones. Therefore, experiments were performed to test if indeed that vagal pathway underlies the effect of fasting. To test if fasting-induced enhancement of nociception requires the vagus nerve, in one group of rats (n⫽6) the subdiaphragmatic branch of the vagus was sectioned two weeks before the beginning of the 48-h fasting period. As shown in Fig. 3A, compared with control (sham surgery) fasted rats (n⫽6), nociceptive response to formalin in vagotomized fasted rats was significantly reduced during the interphase (F(2,15)⫽16.369; P⬍0.001), but not during phase 1 or phase 2. Next, to determine if fasting-induced enhancement of nociception requires the adrenal medulla, adrenal medullectomy was performed in one group of rats (n⫽6). As shown in Fig. 3A, compared with control (sham surgery)fasted rats, the nociceptive response to formalin in adrenal medullectomized fasted rats was significantly reduced during the interphase (n⫽6; P⫽0.132), but not during phase 1 or phase 2. Finally, we investigated the role of gonadal hormones in fasting-induced enhancement of nociception. As shown in Fig. 3B, orchidectomy prevented the fasting-induced enhancement of nociceptive response to formalin, so that there was no significant difference between the response to formalin in fasted (n⫽8) versus non-fasted (n⫽8) female rats. Administration of 17-estradiol to orchidectomized rats (n⫽8), reconstituted the fasting-induced enhancement of the behavioral response to formalin. There were significant differences between the groups during all phases of the formalin test: phase 1 (F(2,21)⫽4.094; P⫽0.032), interphase (F(2,21)⫽27.223; P⬍0.001) and phase 2 (F(2,21)⫽6.103; P⫽0.008). Post hoc tests showed that during phase 1, the orchidectomized, estradiol-treated fasted group was significantly different only from the orchidectomized non-fasted group (P⫽0.04), but during the interphase, the orchidectomized, estradiol-treated fasted group was significantly different from both orchidectomized groups (P⬍0.001 each). Also during phase 2, the orchidectomized, estradiol-treated fasted group was significantly different from the orchidectomized non-fasted (P⫽0.037) and the orchidectomized fasted group (P⫽0.015) groups.
DISCUSSION This study demonstrates that, in female rats, fasting can act as a physiological stimulus of the recently described endogenous nociceptive modulation pathway involving the vagus, adrenal medulla, and gonadal hormones (Khasar et al., 2001). For most organisms existing in the wild, brief periods of fasting are not unusual. This suggests that the
Fig. 3. The effect of fasting in female rats involves the vagus nerve, adrenal medulla and gonadal hormones. (A) Sectioning the vagus nerve significantly reduced fasting-enhanced behavioral responses in the formalin test during the interphase interval. Removal of the adrenal medullae also attenuated the fasting-induced enhancement of responses in the formalin test. Sham abdominal surgery had no significant effect on the effect of fasting. (B) Removal of the ovaries also prevented the fasting-induced enhancement of responses in the formalin test during the interphase. Thus, there were no significant differences in the responses between orchidectomized rats that were fasted and not fasted. The effect of orchidectomy was reversed by administration of 17-estradiol.
pathway functions not merely as a static control over pain sensitivity, but that it is adapted to dynamically modulate nociception. The pro-nociceptive effect of fasting was much greater in female than male rats. This sex-dependence is consistent with the idea that the vagus-dependent nociceptive modulation pathway is tonically active in males but much less active in females (based on the observation that vagotomy increases pain threshold in males but has little effect in females; Khasar et al., 2001). It is possible that under more naturalistic conditions where food is not available at all times ad libitum, females might also exhibit some baseline activation of the system. Under such con-
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ditions, the system might be capable of performing ongoing up- and down-adjustments in nociceptive sensitivity in response to the continually changing feeding status and hormonal levels of the animal. In this regard it would be of interest for future studies to examine the time course and stimulus intensity-response relationship for fasting-induced modulation of nociception. The present data indicate that increases and decreases in vagus-dependent pain enhancement can take place in less than 48 h. Because vagotomy reversed the effect of fasting on nociception, we suggest that enhancement of nociception by fasting may be caused by an increase in vagal afferent activity. Consistent with this idea, it has been reported that vagal activity increases during fasting (Szekely et al., 2000). There is evidence that activity in different vagal afferents can be either pro- or anti-nociceptive (Gebhart and Randich, 1992; Randich and Gebhart, 1992; Watkins et al., 1994). We hypothesize that fasting activates vagal afferents that are pro-nociceptive with respect to formalininduced behaviors. This contrasts with the effect of vagal afferent stimulation on bradykinin-induced hyperalgesia, which we showed previously is anti-nociceptive (Khasar et al., 1998a,b). Although some vagal afferents are glucoreceptors (Paintal, 1973; Mei, 1985), it seems unlikely that these contribute to the effects observed in the present experiments, since hypoglycemia during fasting (Bequet et al., 2000) would tend to decrease activity in these fibers. Previous studies using normal, non-fasted, male rats have reported effects of sucrose ingestion on nociception (Roane and Martin, 1990; Frye et al., 1993; d’Anci et al., 1996; Kanarek et al., 1997, 2001; Mukherjee et al., 2000; Mukherjee et al., 2002), including analgesia in formalininduced nociceptive behaviors (Mukherjee et al., 2000; Dutta et al., 2001). However, our finding that glucose supplementation does not prevent the fasting-induced enhancement of nociception suggests that altered glucose metabolism does not play an essential role in the effect. On the other hand, we found that petrolatum gavage reversed fasting-induced enhancement of nociception (during the interphase interval of the formalin response) while sham gavage had no effect. This reversal might be explained by a gavage-induced decrease of activity in mechanoreceptive vagal afferents (Andrews et al., 1980; Blackshaw et al., 1987; Davison and Clarke, 1988). Specifically, the petrolatum gavage might cause a decrease in activity by triggering the decrease in fasting-induced cyclic motor activity such as typically occurs in the duodenum upon end-of-fast feeding (Kihara et al., 2001). Studies by Watkins and coworkers (Watkins et al., 1994) suggested that vagal activity may play an important role in producing the systemic hyperalgesia associated with illness. However, those investigators found no effect of vagotomy on formalin-induced nociception. This apparent contradiction with our observations probably reflects the use of male rats only by Watkins et al. The interplay between vagal activity, fasting, and nociception in a natural state of sickness may be quite complex, with vagus-dependent hyperalgesia and illness promoting anorexia
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(Konsman and Dantzer, 2001). Our results suggest that anorexia-induced fasting could, in turn, further exacerbate the systemic hyperalgesia associated with illness. Subdiaphragmatic vagal afferents converge on the nucleus of the solitary tract (NTS), which has projections to the paraventricular nuleus (PVN) (Berthoud and Neuhuber, 2000), and the adrenal medulla is innervated by preganglionic sympathetic nerves that receive input from the PVN (Motawei et al., 1999). The PVN plays a very important role in the regulation of feeding, satiety and fasting (Leibowitz, 1988; Maeda et al., 1996) as well as nociception (Snowball et al., 2000; Hallbeck, 2000; Palkovits et al., 1999). We hypothesize that fasting stimulates vagal afferent activity that is transmitted to the NTS, from which it is relayed to the PVN and the adrenal medulla. We have previously shown that vagal modulation of tonic nociception is sexually dimorphic (Khasar et al., 2001), and that adrenal medulla-dependent regulation of the inflammatory response is sexually dimorphic (Green et al., 1999, 2001). Maeda and colleagues have detected estrogen receptors in the NTS and in the PVN, that are up-regulated during fasting (Maeda et al., 1996) and there is evidence that plasma estrogen levels also increase during fasting (Otukonyong et al., 2000). Thus, with presence of estrogen receptors on nodose ganglion (vagal afferent) neurons (Papka et al., 1997, 2001), the NTS, the PVN (Maeda et al., 1996) and the adrenal medulla (Green et al., 1999), the neural pathway proposed to mediate fastinginduced enhancement of formalin-induced nociceptive behaviors is sexually dimorphic at several levels. Although petrolatum gavage prevented the effect of fasting during the interphase, it had no significant effect on the fasting-enhanced behavior during phase 1 or phase 2. It is thought that phase 1 may be caused by increased activity in primary afferent nociceptors due to their direct activation by formalin (Hunskaar and Hole, 1987) as well as by inflammatory mediator-induced nociceptor sensitization (Correa and Calixto, 1993; Doak and Sawynok, 1997; Parada et al., 2001). Phase 2, in addition to increased activity of sensitized nociceptors, may also involve sensitization of nociceptive circuitry in the CNS (Dickenson and Sullivan, 1987; Coderre and Melzack, 1992). The decrease in nociceptive behavior that ordinarily occurs during the interphase, the period between phases 1 and 2, is thought to reflect active inhibition of nociceptive transmission in the spinal cord (Franklin and Abbott, 1993; Kaneko and Hammond, 1997; Omote et al., 1998; Henry et al., 1999). Therefore, we speculate that the decrease in fasting-induced behavior during the interphase interval, caused by petrolatum infusion, may involve a restoration of spinal inhibitory mechanisms that would normally operate during that period. As the circuitry underlying interphase inhibition becomes better understood, it will be of great interest to investigate possible sexual dimorphism in those pathways, as well as interactions with vagal and adrenal mechanisms implicated in the effect of fasting. In summary, we have shown that fasting can act as a physiological stimulus of the endogenous pain-modulation pathway involving the vagus, adrenal medulla, and go-
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nadal hormones. This system may be active during periods of fasting, whether caused by food shortage or perhaps motivated by illness. Furthermore, we can speculate that the system might become particularly important in adjusting pain sensitivity in female rats during the process of reproduction, when gonadal hormones, vagal activity, and nociception are altered. Acknowledgements—The authors are grateful to Drs. Steven Paul and Robert Gear for assistance with statistical analyses, and to Mr. Dennis Mendoza for technical assistance. This research was supported by the National Institutes of Health grant NR04880.
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(Accepted 31 December 2002)