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Central nitric oxide synthase inhibition restores behaviorally mediated lipopolysaccharide induced fever in near-term rats
Physiology & Behavior 94 (2008) 630–634
Contents lists available at ScienceDirect
Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Brief communication
Central nitric oxide synthase inhibition restores behaviorally mediated lipopolysaccharide induced fever in near-term rats Denovan P. Begg a,⁎, Michael L. Mathai b, Michael J. McKinley b, Peter B. Frappell c, Stephen Kent a a b c
School of Psychological Science, La Trobe University, Bundoora, Victoria, 3086 Australia Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Australia Department of Zoology, La Trobe University, Bundoora, Victoria, Australia
A R T I C L E
I N F O
Article history: Received 25 November 2007 Received in revised form 28 March 2008 Accepted 31 March 2008 Keywords: Fever Nitric oxide Pregnant Behavioral thermoregulation Thermal gradient
A B S T R A C T It has recently been established that the febrile response to bacterial endotoxin attenuated late in pregnancy is partially restored by central inhibition of nitric oxide synthase (NOS). To determine if this restoration of the febrile response also extends to warm-seeking behavior, we used a thermocline to allow animals to select their preferred ambient temperature. Near-term pregnant (day 19–20) and aged matched non-pregnant rats were given an I.P. injection of lipopolysaccharide (LPS, 50 μg/kg) and an intracerebroventricular (I.C.V.) injection of an inhibitor of NOS, NG-monomethyl-L-arginine acetate salt (L-NMMA, 100 μg) or vehicle. Core body temperature and self-selected ambient temperature were measured for 6 h after injection. Inhibition of brain NOS before LPS injection resulted in a significant febrile response with an associated increase in selected ambient temperature in both near-term and non-pregnant animals. As expected, near-term dams that received I.C.V. vehicle+I.P. LPS did not have a febrile response but displayed a small hypothermic reaction with no change in selected ambient temperature. We conclude that blockade of brain NOS restores maternal hyperthermic and warm-seeking responses to LPS in near-term pregnancy. © 2008 Elsevier Inc. All rights reserved.
Fever is a regulated increase in core body temperature (Tb) which is the result of the initiation of heat producing and conserving mechanisms. It is an important part of the immuno-physiological response of the host to infection and inflammation, improving the activation of lymphocytes and reducing the replication of many micro-organisms [1]. Investigators have used a range of pyrogenic stimuli, such as administration of lipopolysaccharide (LPS), carrageenin, or turpentine to simulate infection and inflammation. It is well established that the febrile response to infectious stimuli is attenuated late in pregnancy in several mammalian species [2,3]. Further, there are some reports that peripheral injection of LPS at doses which would generally result in fever in normal rats can induce hypothermic responses in near-term pregnant rats [4,5]. This hypothermia appears to be regulated as these animals did not seek warmer ambient temperatures (Ta) when placed in a thermocline [6]. In contrast, non-pregnant rats treated with low doses of LPS develop a fever that can be accompanied by an increase in chosen Ta [7] while at high doses LPS causes hypothermia and cold seeking-behavior [8]. Accompanying the attenuation of fever, near-term pregnant rats injected with LPS, have lower levels of cyclo-oxygenase-2 (COX-2) in the hypothalamus [9] and decreased prostaglandin E2 in their cerebrospinal fluid (CSF) compared to non-pregnant controls [10].
⁎ Corresponding author. Tel.: +61 3 9479 1816; fax: +61 3 9479 1956. E-mail address: [email protected] (D.P. Begg). 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.03.022
Pregnant dams treated with LPS have increased plasma concentrations of the endogenous antipyretic molecule IL-1 receptor antagonist (IL-1ra) [5], but no differences in concentrations of the proinflammatory cytokines IL-1β, IL-6, TNFα, and IFNγ, and an unchanged IL-6 STAT3 signaling pathway when compared to non-pregnant controls [11]. However, following turpentine induced inflammation there is evidence that the IL-6 response is suppressed in near-term pregnant animals [12]. Nitric oxide (NO) is a free radical gas which affects neurotransmission primarily through cGMP formation. Neurons containing nitric oxide synthase (NOS) have been identified in the medial preoptic area of the hypothalamus [13], a key integrative area for thermoregulatory signals, and there is substantial evidence that brain NO is involved in thermoregulation through neural signalling [14]. NO donors cause hypothermic responses when administered peripherally [15] or centrally [16]. Centrally administered NOS inhibitors increase Tb in lightly restrained [14], exercising [17], and LPS injected rats [16] and reduce the hypothermic effects of AVP and insulin [18]. In contrast, peripheral NOS inhibition attenuates fever at a Ta below, but not above, thermoneutrality [19] which indicates this is due to NO increasing vasodilatation. Neuronal NOS is increased in the hypothalamus during the late stages of pregnancy [20,21] and recently we demonstrated that a central injection of the NOS inhibitor NG-monomethyl-L-arginine citrate (L-NMMA) results in the restoration of the febrile response to LPS at this time [22]. Given behavioral and autonomic thermoregu-
D.P. Begg et al. / Physiology & Behavior 94 (2008) 630–634
latory mechanisms are independently controlled [23], the aim of the current study was to determine whether the restored febrile response in near-term pregnant rats following NOS inhibition influences behavioral thermoregulation. It was hypothesized that inhibiting central NOS would restore LPS-induced fever in near-term pregnant rats and this would be accompanied by an increase in chosen Ta. 1. Materials and methods 1.1. Animals housing 24 female Sprague Dawley rats were obtained from Monash SPF animal services (Clayton, Vic, Australia) and housed in the Central Animal House of La Trobe University. All testing was approved by the La Trobe University Animal Ethics Committee (AEC 05/13) under NHMRC guidelines. Animals arrived in the laboratory at 8 weeks of age and were given at least one week to acclimatise before 12 of the rats were mated. They were individually housed, fed standard laboratory rat pellets, and this, along with water, was available ad libitum. The animals were kept in a 12:12 light–dark cycle with the Ta maintained at 25–27 °C. 1.2. Lateral ventricle cannulation and data logger implantation Animals were anaesthetised with an I.P. injection of ketamine (61 mg/kg) and xylazine (9 mg/kg); carpofen (7 mg/kg) was administered S.C. to reduce postoperative discomfort. A 23 gauge cannula was aligned with bregma and moved 1.5 mm to the right and 0.2 mm caudally. A hole was drilled at this point through the skull; the cannula was positioned 3 mm below the dura mater into the lateral ventricle. The position was confirmed by flow of CSF under gravity. The cannula was secured to the skull with dental acrylic and screws and capped to prevent blockage. Anaesthetised animals then had a temperature data logger (Subcue, Calgary, Canada) implanted into the peritoneal cavity via a 1 cm incision in the skin and muscle wall. The muscle and skin were sutured and antibacterial solution and bupivacaine were administered topically. A recovery period of at least 7 days followed the surgery. 1.3. Chemicals
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1.5. Procedure A 12:12-h light–dark photoperiod was maintained with the onset of light at 0700. Experiments were started at 0900 when, rats (day 18– 19 of pregnancy) were placed in the chamber for a 24 h acclimation. At 0900 on day 2 animals (now at day 19–20 of pregnancy) were given either I.C.V. L-NMMA (100 μg) and I.P. LPS (50 μg/kg) or I.C.V. artificial cerebrospinal fluid (aCSF) and I.P. LPS (50 μg/kg). All I.C.V. injections were in 3 μl of aCSF. The injections were given 2 h after lights-on (Time 0). I.C.V. injections were given immediately preceding I.P. injections. Tb and position in the thermal gradient were monitored for the next 5 h. Tb was collected at 5 min intervals by the implanted data logger. The outputs from the photocells were recorded every 5 min using an analog-to-digital converter (PowerLab/800, AD Instruments, NSW, Australia). 1.6. Statistical analyses Selected Ta (calculated from photocell position) was averaged for each 30 min period; Tb was also averaged for 30 min. The groups were compared using a 2 (pregnant/non-pregnant) × 2 (L-NMMA/aCSF) × 14 (time points) repeated measures ANOVA for both Tb and Ta with post hoc protected Least Significant Difference comparisons performed to detect significant differences between groups using Statistica 7. Results are presented as mean ± S.E.M. 2. Results 2.1. Core body temperature The baseline Tb of pregnant rats was 0.5 °C lower than nonpregnant animals (37.0 ± 0.2 °C vs. 37.5 ± 0.2 °C; p b 0.05). LPS injection with aCSF in day 19–20 pregnant dams resulted in a slight hypothermic reaction 120–150 min after injection (see Fig. 1). In contrast, the same treatment in non-pregnant females resulted in a febrile response (2.2 ± 0.2 °C; see Fig. 2). Inhibition of central NOS paired with LPS injection induced equivalent fevers in day 19–20 pregnant dams (1.6 ± 0.3 °C) and nonpregnant females (2.0 ± 0.3 °C). There were no significant differences between the Tb of these 2 groups. In contrast, day 19–20 pregnant
L-NMMA acetate salt (MW 248.3) and LPS extracted from Escherichia coli (serotype 0111:B4) were purchased from Sigma (Castle Hill, NSW, Australia). Ketamine and xylazine were purchased from Ilium Laboratories (Smithfield, NSW, Australia) and carpofen from Allhank Trading Co. (South Melbourne, VIC, Australia). 1.4. Thermal gradient The chamber (1500 mm × 100 mm × 100 mm) consisted of a base and walls constructed from aluminium and fitted with a plastic floor raised 10 mm above the base. The chamber had a Plexiglas lid fitted with an inlet and outlet port; air flow through the chamber was maintained at 1200 ml/min. The chamber was preheated at one end by water at 43 °C and cooled at the other end by water at 5 °C, thereby establishing a thermal gradient that was approximately linear from 15 °C to 35 °C. The sides of the chamber were fitted with a series of infrared phototransistors spaced at 35-mm intervals; the photocell closest to the warm end of the gradient that was obscured by the rat recorded the position (i.e., preferred Ta) of the animal within the thermal gradient, as previously described [24]. Food and water were available ad libitum; the crushed food was distributed along the gradient, water was provided at three sites, centrally and one at each end of the chamber. A single pregnant rat, or an age matched nonpregnant control, was placed in the chamber for determination of its preferred ambient temperature.
Fig. 1. The fever of near-term pregnant rats treated with LPS was attenuated, in response to LPS (○) and a small hypothermic response occurred. Combined treatment with LNMMA + LPS (●) restored the febrile response (Mean Tb ± SEM). ⁎ denotes significant difference from baseline (L-NMMA + LPS group); § denotes significant difference from baseline (LPS group); † denotes significant difference between groups.
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Fig. 2. Non-pregnant female rats displayed a febrile response to LPS (○) treatment or L-NMMA + LPS treatment (●) (Mean Tb ± SEM). ⁎ denotes significant difference from baseline (L-NMMA + LPS group); § denotes significant difference from baseline (LPS group).
dams that received LPS alone had a significantly lower Tb than the animals that had also received L-NMMA from the 60 min time point on (all p b 0.05). 2.2. Chosen ambient temperature Chosen Ta at baseline was not significantly different between pregnant and non-pregnant rats. Injection of LPS and aCSF led to an increase in chosen Ta in non-pregnant animals beginning at 90 min, with a maximal increase of 2.9 ± 0.8 °C at the 330–360 min time point. There was no change in the chosen temperature of pregnant rats treated with LPS and aCSF. Injection of LPS and L-NMMA in pregnant (Fig. 3) and nonpregnant (Fig. 4) rats led to similar increases in chosen Ta with a maximal increases occurring at the 120–150 min time point for
Fig. 3. Near-term pregnant rats treated with LPS did not alter Ta in response to LPS (○). Combined treatment with L-NMMA + LPS (●) resulted in increases in chosen Ta at 8 out of ten time points 90 min after injection (Mean Ta ± SEM). ⁎ denotes significant difference from baseline (L-NMMA + LPS group); † denotes significant difference between groups.
Fig. 4. Non-pregnant female rats treated with LPS increased Ta in response to LPS (○) treatment or L-NMMA + LPS treatment (●) at all time points after 120 min (Mean Ta ± SEM). ⁎ denotes significant difference from baseline (L-NMMA + LPS group); § denotes significant difference from baseline (LPS group).
pregnant rats (2.7 ± 1.1 °C) and the 90–120 min time point for nonpregnant (2.9 ± 1.2 °C). 3. Discussion Our data demonstrate that blockade of brain nitric oxide production by L-NMMA injection restores warm-seeking behavior in nearterm pregnant rats. A similar increase in self-selected Ta was observed in non-pregnant animals administered LPS as was observed in pregnant animals injected with LPS and L-NMMA. The increase in chosen Ta confirms that the increased Tb observed in the near-term pregnant animals treated in combination with the NOS inhibitor and LPS, in both this study and previously [22], is indeed a febrile response. While the large increase in preferred environmental temperature was sustained for about 3 h in the pregnant rat, these animals subsequently returned to slightly lower environmental temperatures, which did not occur in non-pregnant animals. This later decrease in Ta could be the result of a reduction in the inhibition of NOS due to clearance of the pharmacological antagonist. The increase of chosen Ta is a well established phenomenon in rats injected with LPS to induce fever [25,7] and the heat seeking behavior following LPS administration in the current study was similar to that seen in previous reports [26,25]. The baseline temperature of pregnant animals was significantly lower than non-pregnant animals, a finding that has been reported previously [4], and has been found to be influenced by central administration of the angiotensin (AT)-1 receptor antagonist candesartan [27]. Pregnant animals injected with LPS alone showed a small but distinct pattern of hypothermia, as has previously been reported [4]. There was no change in chosen Ta in these animals as they became hypothermic, consistent with previous findings [6]. There was a clear biphasic fever in both non-pregnant groups treated with LPS alone or in combination with L-NMMA. Pregnant animals injected with L-NMMA and LPS produced a fever slightly smaller than non-pregnant animals and Tb did not increase into a second phase as it did in nonpregnant animals. Interestingly, this coincided with a decrease in the preferred Ta from the peak at 120–180 min. This may reflect a reduction in NOS inhibition over time, with L-NMMA having an in vivo half-life of around 75 min [28]. As with our previous report NOS inhibition did not affect the LPS induced fever in non-pregnant animals [22].
D.P. Begg et al. / Physiology & Behavior 94 (2008) 630–634
The increase in central NO in near-term pregnancy [20] appears to be due to increased oxytocinergic cell signalling [21]. The attenuation of fever near-term may either be the result of the hormonal state during late pregnancy or an evolutionary mechanism to protect the fetus. However, given that the increase in NOS appears to be present during lactation in some regions of the brain [21], a period when fever can be induced, the attenuation may be due to site specific changes in NO. The warm-seeking behavior and autonomically-mediated thermogenic activity appear to be controlled by different sites in the brain. For example the preoptic area (POA) appears to be essential for the autonomic response in fever, as animals with a lesion of the POA are unable to increase their core temperature in response to PGE [29]. However, when the animals were placed in a thermocline the warmseeking behavioral response to LPS was intact, and they moved to a warmer area to increase core Tb, generating fever [7]. Our findings indicate that, NO inhibition restores both thermogenic and thermoadaptive behavioral responses to LPS. The neurophysiology of heatseeking behavior is not well understood. Lateral, but not anterior, hypothalamic lesion leads to a loss of behavioral regulation [30,31] and while the paraventricular nucleus (PVN) and dorsomedial nucleus (DMN) are involved in cold-seeking behavior, they are not involved in heat-seeking behavior [7]. NO inhibits COX-2 mediated PGE2 activity[32], therefore, the increased NOS in the brain during pregnancy may be responsible for the reduction of COX-2 [9] and PGE2 [10]. This could be the result of NO inhibiting COX-2 activity by nitrosylation of a tyramine in the COX catalytic site [33] and/or by removing the inhibitory effect of excess NO on noradrenaline [34]. However, fever is attenuated during pregnancy even when PGs are injected directly [35] and it has been proposed previously that the attenuation of fever in pregnancy is independent of COX-2 [36]. PGs reduce cAMP levels and low levels of both cGMP and cAMP have been implicated in the generation of fever [37], while there is evidence that NO inhibits pyrogenic mechanisms by increasing cGMP levels in the preoptic area [16]. In the near-term animals, NO [20,38,21] and presumably cGMP activity is increased, thereby inhibiting normal pyrogenic signalling. Therefore, it is likely that the effects on fever are due to the opposite effects of NO and PGs on cGMP signalling. Inhibition of NOS blocks formation of cGMP, returning the activity of brain regions involved in thermogenic and thermo-adaptive behaviors to the non-pregnant state. The effect of the establishment of fever in dams on the offspring is unknown; however, it would increase the likelihood of heat stress harming the fetus. Furthermore, immune activation during pregnancy has been linked to hypothalamic and hippocampal abnormalities [39,40] cognitive and psychiatric deficits [40,41]. NO may act as a protective antipyretic agent during late pregnancy to prevent injurious outcomes in the offspring. Acknowledgments M. L. Mathai was supported by NHMRC of Australia Project Grant 232306, the G. Harold and Leila Y. Mathers Trust, and the Robert J. Kleberg Jr. and Helen C. Kleberg Foundation.
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Contents lists available at ScienceDirect
Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Brief communication
Central nitric oxide synthase inhibition restores behaviorally mediated lipopolysaccharide induced fever in near-term rats Denovan P. Begg a,⁎, Michael L. Mathai b, Michael J. McKinley b, Peter B. Frappell c, Stephen Kent a a b c
School of Psychological Science, La Trobe University, Bundoora, Victoria, 3086 Australia Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Australia Department of Zoology, La Trobe University, Bundoora, Victoria, Australia
A R T I C L E
I N F O
Article history: Received 25 November 2007 Received in revised form 28 March 2008 Accepted 31 March 2008 Keywords: Fever Nitric oxide Pregnant Behavioral thermoregulation Thermal gradient
A B S T R A C T It has recently been established that the febrile response to bacterial endotoxin attenuated late in pregnancy is partially restored by central inhibition of nitric oxide synthase (NOS). To determine if this restoration of the febrile response also extends to warm-seeking behavior, we used a thermocline to allow animals to select their preferred ambient temperature. Near-term pregnant (day 19–20) and aged matched non-pregnant rats were given an I.P. injection of lipopolysaccharide (LPS, 50 μg/kg) and an intracerebroventricular (I.C.V.) injection of an inhibitor of NOS, NG-monomethyl-L-arginine acetate salt (L-NMMA, 100 μg) or vehicle. Core body temperature and self-selected ambient temperature were measured for 6 h after injection. Inhibition of brain NOS before LPS injection resulted in a significant febrile response with an associated increase in selected ambient temperature in both near-term and non-pregnant animals. As expected, near-term dams that received I.C.V. vehicle+I.P. LPS did not have a febrile response but displayed a small hypothermic reaction with no change in selected ambient temperature. We conclude that blockade of brain NOS restores maternal hyperthermic and warm-seeking responses to LPS in near-term pregnancy. © 2008 Elsevier Inc. All rights reserved.
Fever is a regulated increase in core body temperature (Tb) which is the result of the initiation of heat producing and conserving mechanisms. It is an important part of the immuno-physiological response of the host to infection and inflammation, improving the activation of lymphocytes and reducing the replication of many micro-organisms [1]. Investigators have used a range of pyrogenic stimuli, such as administration of lipopolysaccharide (LPS), carrageenin, or turpentine to simulate infection and inflammation. It is well established that the febrile response to infectious stimuli is attenuated late in pregnancy in several mammalian species [2,3]. Further, there are some reports that peripheral injection of LPS at doses which would generally result in fever in normal rats can induce hypothermic responses in near-term pregnant rats [4,5]. This hypothermia appears to be regulated as these animals did not seek warmer ambient temperatures (Ta) when placed in a thermocline [6]. In contrast, non-pregnant rats treated with low doses of LPS develop a fever that can be accompanied by an increase in chosen Ta [7] while at high doses LPS causes hypothermia and cold seeking-behavior [8]. Accompanying the attenuation of fever, near-term pregnant rats injected with LPS, have lower levels of cyclo-oxygenase-2 (COX-2) in the hypothalamus [9] and decreased prostaglandin E2 in their cerebrospinal fluid (CSF) compared to non-pregnant controls [10].
⁎ Corresponding author. Tel.: +61 3 9479 1816; fax: +61 3 9479 1956. E-mail address: [email protected] (D.P. Begg). 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.03.022
Pregnant dams treated with LPS have increased plasma concentrations of the endogenous antipyretic molecule IL-1 receptor antagonist (IL-1ra) [5], but no differences in concentrations of the proinflammatory cytokines IL-1β, IL-6, TNFα, and IFNγ, and an unchanged IL-6 STAT3 signaling pathway when compared to non-pregnant controls [11]. However, following turpentine induced inflammation there is evidence that the IL-6 response is suppressed in near-term pregnant animals [12]. Nitric oxide (NO) is a free radical gas which affects neurotransmission primarily through cGMP formation. Neurons containing nitric oxide synthase (NOS) have been identified in the medial preoptic area of the hypothalamus [13], a key integrative area for thermoregulatory signals, and there is substantial evidence that brain NO is involved in thermoregulation through neural signalling [14]. NO donors cause hypothermic responses when administered peripherally [15] or centrally [16]. Centrally administered NOS inhibitors increase Tb in lightly restrained [14], exercising [17], and LPS injected rats [16] and reduce the hypothermic effects of AVP and insulin [18]. In contrast, peripheral NOS inhibition attenuates fever at a Ta below, but not above, thermoneutrality [19] which indicates this is due to NO increasing vasodilatation. Neuronal NOS is increased in the hypothalamus during the late stages of pregnancy [20,21] and recently we demonstrated that a central injection of the NOS inhibitor NG-monomethyl-L-arginine citrate (L-NMMA) results in the restoration of the febrile response to LPS at this time [22]. Given behavioral and autonomic thermoregu-
D.P. Begg et al. / Physiology & Behavior 94 (2008) 630–634
latory mechanisms are independently controlled [23], the aim of the current study was to determine whether the restored febrile response in near-term pregnant rats following NOS inhibition influences behavioral thermoregulation. It was hypothesized that inhibiting central NOS would restore LPS-induced fever in near-term pregnant rats and this would be accompanied by an increase in chosen Ta. 1. Materials and methods 1.1. Animals housing 24 female Sprague Dawley rats were obtained from Monash SPF animal services (Clayton, Vic, Australia) and housed in the Central Animal House of La Trobe University. All testing was approved by the La Trobe University Animal Ethics Committee (AEC 05/13) under NHMRC guidelines. Animals arrived in the laboratory at 8 weeks of age and were given at least one week to acclimatise before 12 of the rats were mated. They were individually housed, fed standard laboratory rat pellets, and this, along with water, was available ad libitum. The animals were kept in a 12:12 light–dark cycle with the Ta maintained at 25–27 °C. 1.2. Lateral ventricle cannulation and data logger implantation Animals were anaesthetised with an I.P. injection of ketamine (61 mg/kg) and xylazine (9 mg/kg); carpofen (7 mg/kg) was administered S.C. to reduce postoperative discomfort. A 23 gauge cannula was aligned with bregma and moved 1.5 mm to the right and 0.2 mm caudally. A hole was drilled at this point through the skull; the cannula was positioned 3 mm below the dura mater into the lateral ventricle. The position was confirmed by flow of CSF under gravity. The cannula was secured to the skull with dental acrylic and screws and capped to prevent blockage. Anaesthetised animals then had a temperature data logger (Subcue, Calgary, Canada) implanted into the peritoneal cavity via a 1 cm incision in the skin and muscle wall. The muscle and skin were sutured and antibacterial solution and bupivacaine were administered topically. A recovery period of at least 7 days followed the surgery. 1.3. Chemicals
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1.5. Procedure A 12:12-h light–dark photoperiod was maintained with the onset of light at 0700. Experiments were started at 0900 when, rats (day 18– 19 of pregnancy) were placed in the chamber for a 24 h acclimation. At 0900 on day 2 animals (now at day 19–20 of pregnancy) were given either I.C.V. L-NMMA (100 μg) and I.P. LPS (50 μg/kg) or I.C.V. artificial cerebrospinal fluid (aCSF) and I.P. LPS (50 μg/kg). All I.C.V. injections were in 3 μl of aCSF. The injections were given 2 h after lights-on (Time 0). I.C.V. injections were given immediately preceding I.P. injections. Tb and position in the thermal gradient were monitored for the next 5 h. Tb was collected at 5 min intervals by the implanted data logger. The outputs from the photocells were recorded every 5 min using an analog-to-digital converter (PowerLab/800, AD Instruments, NSW, Australia). 1.6. Statistical analyses Selected Ta (calculated from photocell position) was averaged for each 30 min period; Tb was also averaged for 30 min. The groups were compared using a 2 (pregnant/non-pregnant) × 2 (L-NMMA/aCSF) × 14 (time points) repeated measures ANOVA for both Tb and Ta with post hoc protected Least Significant Difference comparisons performed to detect significant differences between groups using Statistica 7. Results are presented as mean ± S.E.M. 2. Results 2.1. Core body temperature The baseline Tb of pregnant rats was 0.5 °C lower than nonpregnant animals (37.0 ± 0.2 °C vs. 37.5 ± 0.2 °C; p b 0.05). LPS injection with aCSF in day 19–20 pregnant dams resulted in a slight hypothermic reaction 120–150 min after injection (see Fig. 1). In contrast, the same treatment in non-pregnant females resulted in a febrile response (2.2 ± 0.2 °C; see Fig. 2). Inhibition of central NOS paired with LPS injection induced equivalent fevers in day 19–20 pregnant dams (1.6 ± 0.3 °C) and nonpregnant females (2.0 ± 0.3 °C). There were no significant differences between the Tb of these 2 groups. In contrast, day 19–20 pregnant
L-NMMA acetate salt (MW 248.3) and LPS extracted from Escherichia coli (serotype 0111:B4) were purchased from Sigma (Castle Hill, NSW, Australia). Ketamine and xylazine were purchased from Ilium Laboratories (Smithfield, NSW, Australia) and carpofen from Allhank Trading Co. (South Melbourne, VIC, Australia). 1.4. Thermal gradient The chamber (1500 mm × 100 mm × 100 mm) consisted of a base and walls constructed from aluminium and fitted with a plastic floor raised 10 mm above the base. The chamber had a Plexiglas lid fitted with an inlet and outlet port; air flow through the chamber was maintained at 1200 ml/min. The chamber was preheated at one end by water at 43 °C and cooled at the other end by water at 5 °C, thereby establishing a thermal gradient that was approximately linear from 15 °C to 35 °C. The sides of the chamber were fitted with a series of infrared phototransistors spaced at 35-mm intervals; the photocell closest to the warm end of the gradient that was obscured by the rat recorded the position (i.e., preferred Ta) of the animal within the thermal gradient, as previously described [24]. Food and water were available ad libitum; the crushed food was distributed along the gradient, water was provided at three sites, centrally and one at each end of the chamber. A single pregnant rat, or an age matched nonpregnant control, was placed in the chamber for determination of its preferred ambient temperature.
Fig. 1. The fever of near-term pregnant rats treated with LPS was attenuated, in response to LPS (○) and a small hypothermic response occurred. Combined treatment with LNMMA + LPS (●) restored the febrile response (Mean Tb ± SEM). ⁎ denotes significant difference from baseline (L-NMMA + LPS group); § denotes significant difference from baseline (LPS group); † denotes significant difference between groups.
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Fig. 2. Non-pregnant female rats displayed a febrile response to LPS (○) treatment or L-NMMA + LPS treatment (●) (Mean Tb ± SEM). ⁎ denotes significant difference from baseline (L-NMMA + LPS group); § denotes significant difference from baseline (LPS group).
dams that received LPS alone had a significantly lower Tb than the animals that had also received L-NMMA from the 60 min time point on (all p b 0.05). 2.2. Chosen ambient temperature Chosen Ta at baseline was not significantly different between pregnant and non-pregnant rats. Injection of LPS and aCSF led to an increase in chosen Ta in non-pregnant animals beginning at 90 min, with a maximal increase of 2.9 ± 0.8 °C at the 330–360 min time point. There was no change in the chosen temperature of pregnant rats treated with LPS and aCSF. Injection of LPS and L-NMMA in pregnant (Fig. 3) and nonpregnant (Fig. 4) rats led to similar increases in chosen Ta with a maximal increases occurring at the 120–150 min time point for
Fig. 3. Near-term pregnant rats treated with LPS did not alter Ta in response to LPS (○). Combined treatment with L-NMMA + LPS (●) resulted in increases in chosen Ta at 8 out of ten time points 90 min after injection (Mean Ta ± SEM). ⁎ denotes significant difference from baseline (L-NMMA + LPS group); † denotes significant difference between groups.
Fig. 4. Non-pregnant female rats treated with LPS increased Ta in response to LPS (○) treatment or L-NMMA + LPS treatment (●) at all time points after 120 min (Mean Ta ± SEM). ⁎ denotes significant difference from baseline (L-NMMA + LPS group); § denotes significant difference from baseline (LPS group).
pregnant rats (2.7 ± 1.1 °C) and the 90–120 min time point for nonpregnant (2.9 ± 1.2 °C). 3. Discussion Our data demonstrate that blockade of brain nitric oxide production by L-NMMA injection restores warm-seeking behavior in nearterm pregnant rats. A similar increase in self-selected Ta was observed in non-pregnant animals administered LPS as was observed in pregnant animals injected with LPS and L-NMMA. The increase in chosen Ta confirms that the increased Tb observed in the near-term pregnant animals treated in combination with the NOS inhibitor and LPS, in both this study and previously [22], is indeed a febrile response. While the large increase in preferred environmental temperature was sustained for about 3 h in the pregnant rat, these animals subsequently returned to slightly lower environmental temperatures, which did not occur in non-pregnant animals. This later decrease in Ta could be the result of a reduction in the inhibition of NOS due to clearance of the pharmacological antagonist. The increase of chosen Ta is a well established phenomenon in rats injected with LPS to induce fever [25,7] and the heat seeking behavior following LPS administration in the current study was similar to that seen in previous reports [26,25]. The baseline temperature of pregnant animals was significantly lower than non-pregnant animals, a finding that has been reported previously [4], and has been found to be influenced by central administration of the angiotensin (AT)-1 receptor antagonist candesartan [27]. Pregnant animals injected with LPS alone showed a small but distinct pattern of hypothermia, as has previously been reported [4]. There was no change in chosen Ta in these animals as they became hypothermic, consistent with previous findings [6]. There was a clear biphasic fever in both non-pregnant groups treated with LPS alone or in combination with L-NMMA. Pregnant animals injected with L-NMMA and LPS produced a fever slightly smaller than non-pregnant animals and Tb did not increase into a second phase as it did in nonpregnant animals. Interestingly, this coincided with a decrease in the preferred Ta from the peak at 120–180 min. This may reflect a reduction in NOS inhibition over time, with L-NMMA having an in vivo half-life of around 75 min [28]. As with our previous report NOS inhibition did not affect the LPS induced fever in non-pregnant animals [22].
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The increase in central NO in near-term pregnancy [20] appears to be due to increased oxytocinergic cell signalling [21]. The attenuation of fever near-term may either be the result of the hormonal state during late pregnancy or an evolutionary mechanism to protect the fetus. However, given that the increase in NOS appears to be present during lactation in some regions of the brain [21], a period when fever can be induced, the attenuation may be due to site specific changes in NO. The warm-seeking behavior and autonomically-mediated thermogenic activity appear to be controlled by different sites in the brain. For example the preoptic area (POA) appears to be essential for the autonomic response in fever, as animals with a lesion of the POA are unable to increase their core temperature in response to PGE [29]. However, when the animals were placed in a thermocline the warmseeking behavioral response to LPS was intact, and they moved to a warmer area to increase core Tb, generating fever [7]. Our findings indicate that, NO inhibition restores both thermogenic and thermoadaptive behavioral responses to LPS. The neurophysiology of heatseeking behavior is not well understood. Lateral, but not anterior, hypothalamic lesion leads to a loss of behavioral regulation [30,31] and while the paraventricular nucleus (PVN) and dorsomedial nucleus (DMN) are involved in cold-seeking behavior, they are not involved in heat-seeking behavior [7]. NO inhibits COX-2 mediated PGE2 activity[32], therefore, the increased NOS in the brain during pregnancy may be responsible for the reduction of COX-2 [9] and PGE2 [10]. This could be the result of NO inhibiting COX-2 activity by nitrosylation of a tyramine in the COX catalytic site [33] and/or by removing the inhibitory effect of excess NO on noradrenaline [34]. However, fever is attenuated during pregnancy even when PGs are injected directly [35] and it has been proposed previously that the attenuation of fever in pregnancy is independent of COX-2 [36]. PGs reduce cAMP levels and low levels of both cGMP and cAMP have been implicated in the generation of fever [37], while there is evidence that NO inhibits pyrogenic mechanisms by increasing cGMP levels in the preoptic area [16]. In the near-term animals, NO [20,38,21] and presumably cGMP activity is increased, thereby inhibiting normal pyrogenic signalling. Therefore, it is likely that the effects on fever are due to the opposite effects of NO and PGs on cGMP signalling. Inhibition of NOS blocks formation of cGMP, returning the activity of brain regions involved in thermogenic and thermo-adaptive behaviors to the non-pregnant state. The effect of the establishment of fever in dams on the offspring is unknown; however, it would increase the likelihood of heat stress harming the fetus. Furthermore, immune activation during pregnancy has been linked to hypothalamic and hippocampal abnormalities [39,40] cognitive and psychiatric deficits [40,41]. NO may act as a protective antipyretic agent during late pregnancy to prevent injurious outcomes in the offspring. Acknowledgments M. L. Mathai was supported by NHMRC of Australia Project Grant 232306, the G. Harold and Leila Y. Mathers Trust, and the Robert J. Kleberg Jr. and Helen C. Kleberg Foundation.
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