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Lipopolysaccharide-induced fever alters Hering–Breuer reflex in anesthetized rats
Journal of Thermal Biology 37 (2012) 475–478
Contents lists available at SciVerse ScienceDirect
Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Short Communication
Lipopolysaccharide-induced fever alters Hering–Breuer reflex in anesthetized rats I. Zila, D. Mokra, M. Javorka, K. Javorka, A. Calkovska n Department of Physiology, Jessenius Faculty of Medicine, Comenius University, Mala Hora 4, 036 01 Martin, Slovakia
a r t i c l e i n f o
abstract
Article history: Received 14 December 2011 Accepted 27 April 2012 Available online 8 May 2012
The aim of the study was to test the hypothesis that control of breathing via Hering–Breuer reflex (HB) is influenced by lipopolysaccharide (LPS)-induced fever in rats. Animals were injected intraperitoneally with LPS and control group with an equivalent volume of saline. HB reflex was elicited by inflations of the lungs followed by occlusion of the airways under control of esophageal pressure. Duration of HB reflex (Tapnoe) was continuously reduced as body temperature rose during experiment. Compared to normothermic controls, animals with fever had significant shortening of Tapnoe at 240 min and 300 min after LPS administration. Fever was further accompanied by a reduction in the strength of HB reflex (inhibitory ratio, IR). In comparison with controls, significant decrease of IR was observed at 300 min after LPS injection. Conclusion: altered neural control of breathing demonstrated by decreased power of Hering–Breuer inflation reflex in conditions of LPS-induced fever may facilitate thermal tachypnoea and/or play a role in the origin of respiratory instability accompanying febrile response. & 2012 Published by Elsevier Ltd.
Keywords: Control of breathing Hering–Breuer reflex Lipopolysaccharide Fever Rat model
1. Introduction The control in body temperature (BT) interacts with control of breathing and thus elevation in BT induces respiratory and cardiovascular changes both in humans and animals. Cardiorespiratory responses have been extensively studied in conditions of exogenous increase in core temperature (e.g. Brozmanova et al., 2006; Zila et al., 2007; Rubini, 2011), but rarely in fever. Investigation of respiratory reflexes may serve as a tool to assess neural control of breathing. Hering–Breuer (HB) reflex is elicited by activation of pulmonary slowly adapting stretch receptors and it leads to inhibition of inspiration and prolongation of expiration in order to prevent lung overinflation (Korpas and Tomori, 1979). HB reflex was considered to be a control mechanism with relatively low flexibility, however, recent studies indicate its possible modulation under specific conditions (Siniaia et al., 2000). Power of Hering–Breuer reflex is influenced mainly by chemical control of breathing and metabolic rate (Merazzi and Mortola, 1999) while both these factors are sensitive to body temperature. Moreover, vagal receptors themselves show temperature-sensitivity (Trippenbach, 2001).
n
Corresponding author. Tel.: þ421 43 2633422. E-mail addresses: [email protected] (I. Zila), [email protected] (D. Mokra), [email protected] (M. Javorka), [email protected] (K. Javorka), [email protected] (A. Calkovska). 0306-4565/$ - see front matter & 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jtherbio.2012.04.008
Taken together, increase in BT modifies breathing pattern and contributes to respiratory instability in thermal stress (Voss et al., 2004). The effects of changing vagal neural mechanisms in endotoxin-induced fever are unknown and the pathways mediating thermoregulatory influences on respiratory activity remain to be investigated (Morrison and Nakamura, 2011). Therefore, the aim of the study was to investigate the effect of fever induced by administration of lipopolysaccharide on Hering–Breuer reflex in anesthetized rats and to test the hypothesis that fever significantly affects vagally mediated ventilatory responses.
2. Materials and methods 2.1. Experimental animals and procedures 2.1.1. Animals The experiments were performed on 18 adult male rats (Wistar) with a mean body mass of 34776 g (mean 7SEM).
2.1.2. Material LPS: purified lyophilized phenol extract of Escherichia coli (026:B6, Sigma) was dissolved in sterile saline and frozen in aliquots. Before use, the LPS was diluted in sterile saline and injected intraperitoneally.
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I. Zila et al. / Journal of Thermal Biology 37 (2012) 475–478
2.2. Design of the study The study was performed with accordance to EU Directive 2010/63/EU for animal experiments and approved by the Local Ethics Committee of the Jessenius Faculty of Medicine, Comenius University in Martin. The animals were anaesthetized with a mixture of 600 mg kg 1 urethane and 60 mg kg 1 a-chloralose intraperitoneally. They were tracheotomized and breathed spontaneously through a tracheal tube. The airflow was recorded through the Fleisch head of a pneumotachograph connected to the tracheal tube and tidal volume (VT) was obtained by electronic integration of the airflow signal (ADInstruments Pty Ltd.). The esophageal catheter was placed to record esophageal pressure during ventilation. The polyethylene catheter inserted into the femoral artery was used for arterial blood pressure monitoring and blood samples withdrawal. Blood gases ðPaO2 , PaCO2 Þ and pH analysis was performed using a blood gases analyzer (Rapidlab; Bayer Diagnostics, Germany) and values were corrected for actual body temperature. Total antioxidant status (TAS) was quantified in plasma using ABTS radical formation kinetics (Randox TAS kit, Randox Laboratories Ltd., UK). Differential white blood cell count was determined microscopically after staining by Pappenheim in peripheral blood before and 5 h after LPS/saline instillation. Colonic body temperature (Tc) was continuously measured with thermocouple (MiniLogger) inserted 4–5 cm into the anus. 2.3. Experimental protocol The animals were randomly divided into two groups. Animals of LPS group (n ¼9) received intraperitoneally LPS (100 mg kg 1) and control group (n¼9) received an equivalent volume of sterile saline. All recordings were done before (base) and 60, 120, 180, 240 and 300 min after LPS or saline administration. Hering–Breuer (HB) reflex was elicited by inflations of the lungs followed by occlusion of the airways. Inflation volume was set as 1.5x of mean tidal volume that was estimated from 10 breaths before each test. Inflations were performed at the end of inspiratory phase of breathing cycle (Fig. 1) and controlled by changes in esophageal pressure. Occlusion was maintained until first inspiratory effort occurred as indicated by changes in esophageal pressure. The duration of Hering–Breuer reflex (Tapnoe) was determined as the total time from the beginning of the expiration immediately before the lung inflation to the onset of the next inspiration during the maintained inflation. The strength of Hering–Breuer reflex was quantified as inhibitory ratio (IR) and was calculated as the ratio between Tapnoe and average expiratory time of five breaths preceding the lung inflation (according to Merazzi and Mortola (1999)). 2.4. Statistical analysis The statistical software package SYSTAT 6.0.1 (SPSS Inc., 1996) was used for data analysis. Data were tested for normality of
distribution using Kolmogorov–Smirnov test. Between-group differences were analyzed using Mann–Whitney test, and withingroup differences with Wilcoxon test. Data are expressed as means7S.E.M., differences are considered significant when Po0.05.
3. Results Administration of lipopolysaccharide was accompanied by continuous rise in colonic body temperature compared to salinetreated controls. The intraperitoneal injection of 100 mg kg 1 induced monophasic thermogenic reaction in all animals with peak registered 300 min after LPS administration (Fig. 2). There were no differences in initial values of neutrophil count in the peripheral blood in control vs. LPS groups. Administration of endotoxin evoked systemic inflammatory response accompanied by an increase in neutrophil count at the end of the experiments: 73.3710.8% in LPS group vs. 50.4719.1% in controls; Po0.01. Total antioxidant status was significantly lower in LPS-treated group in comparison with controls (0.86270.082 mmol/l vs.1.0097 0.15 mmol/l; Po0.05). Data on duration and strength of Hering–Breuer reflex are summarized in Figs. 3 and 4. The duration of HB reflex was continuously reduced as body temperature rose during experiment. In comparison with normothermic controls, LPS animals exhibited significant shortening of Tapnoe at 240 min after LPS administration—Tapnoe240: 2.270.3 s vs. 3.470.3 s, Po0.05, as well as at the maximum temperature of 38.6 1C at 300 min after endotoxin injection—Tapnoe300: 1.670.2 s vs. 3.570.3 s, P o0.01 (Fig. 3). Progression of fever response was accompanied by the reduction in the strength of Hering–Breuer reflex (Fig. 4). Inhibitory ratio (IR) was reduced at 180, 240 and 300 min after lipopolysaccharide administration compared to baseline. In comparison with controls, significant decrease of IR was observed at 300 min from LPS injection: 5.2 70.2 vs. 9.2 70.3; Po0.01. The initial values of respiratory parameters did not differ between the groups. In controls VT before and after experiment was unchanged (1.9 70.3 mL vs. 1.870.2 mL; P40.05 ) as was the respiratory rate (97711 breaths per minute; bpm vs. 103714 bpm; P40.05). In LPS group, minute ventilation progressively increased with elevation of body temperature as a result of significant increase in respiratory rate from baseline 105711 bpm to 258719 bpm (Po0.01) at 300 min after LPS administration. Tidal volume in LPS animals was reduced to 1.270.2 mL (at Tc300) in comparison with baseline value (1.870.3 mL, Po0.05). No significant differences were present in absolute values of PaO2 , PaCO2 and pH between LPS and control groups or in values compared to baseline. At 300 min after LPS administration PaCO2 tended to decrease but the drop was not significant (data not shown).
Fig. 1. Record of airflow during inflation of the lungs in a single experiment. Te—duration of expiration, Tapnoe—duration of Hering–Breuer reflex, V —airflow, sec—second.
I. Zila et al. / Journal of Thermal Biology 37 (2012) 475–478
Fig. 2. Colonic body temperature (oC) measured before LPS/saline administration (Tcbase) and 60, 120, 180, 240 and 300 min after LPS/saline administration P o0.05.
Fig. 3. Duration (Tapnoe) of HB reflex measured before LPS/saline administration (Tcbase) and 60, 120, 180, 240 and 300 min after LPS/saline administration. Values are expressed as mean 7SEM. Within-group comparison to baseline (before LPS/ saline injection), # Po 0.05; between-group comparison, *P o 0.05, **P o 0.01.
Fig. 4. Strength (inhibitory ratio, IR) of HB reflex measured before LPS/saline administration (Tcbase) and 60, 120, 180, 240 and 300 min after LPS/saline administration. Values are expressed as mean 7 SEM. Within-group comparison to baseline (before LPS/saline injection), # Po 0.05; between-group comparison, **P o 0.01.
4. Discussion Lipopolysaccharide (LPS) derived from the cell wall of Gramnegative bacteria can evoke fever and systemic inflammatory response. Fever is thought to provide an optimal hyperthermic environment for mounting host defense against invading bacteria and viruses (Morrison and Nakamura, 2011). In this study, thermogenic response in LPS-injected animals was accompanied by increase in neutrophil count in peripheral
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blood and by reduction in total antioxidant status as a consequence of production of wide spectrum of inflammatory mediators and reactive oxygen species (ROS) (Werner et al., 2003; Sebai et al., 2009). The body temperature-induced changes in breathing pattern are evoked by alterations in the peripheral temperature, or modified by shifts in the central body temperature, eventually by other mechanisms. Hyperpnoea associated with elevated core temperature was described for the first time by Haldane (1905) and since then numerous studies have been focused on investigation of breathing control in elevated-body temperature conditions. In some species, a rise in body temperature develops a specialized breathing pattern known as thermal tachypnoea that is mediated by the thermoregulatory system in the preoptic area of the hypothalamus. Thermal tachypnoea can, in some mammals and birds, be controlled by central mechanisms alone, while in others it depends on extrinsic stimuli mediated by way of the vagus nerve (Richards, 1968). During this pattern of ventilation, with an elevated functional residual capacity, a high frequency of breathing and a reduced tidal volume, the upper airways are preferentially ventilated and therefore blood gases and pH are maintained in a physiological range (White, 2006). In our experiments, increase in BT in rats with fever was characterized by rapid shallow breathing with significant increase in respiratory rate and decrease in tidal volumes typical for thermal tachypnoea. PaCO2 in LPS animals tended to decrease suggesting increased ventilation that is largely confined to the dead space with only a small increase in alveolar ventilation (Robertshaw, 2006). In these conditions HB inflation reflex was reduced in its duration and strength. Although mechanisms that elicit a rise in body temperature in exogenous increase in core temperature and fever are different, the results are in accordance with previous study of our group in hyperthermic rabbits (Javorka et al., 1996). However, temperature can also directly affect the activity of the respiratory neural network located in the medulla. For ¨ instance, area of pre-Botzinger complex was suggested to play critical role in a change of breathing pattern during thermal stress (Tryba and Ramirez, 2004). The magnitude of the reflex inhibition of ventilation during sustained inflation is probably determined at different levels within the loop Hering–Breuer reflex. Slowly adapting stretch receptors, chemoreceptors and the central effectiveness and processing of vagal inputs seem to be most important as all these components are sensitive to changes in temperature (Merazzi and Mortola, 1999; Zila et al., 2007) and thus an altered sensitivity could play a key role in modulation of vagally mediated ventilatory responses. Particularly, interaction between chemosensitivity and temperature is suggested to have stronger influence on breathing than the thermogenic inhibition of vagal ventilatory mechanisms and the change in HB reflex in febrile rats could result from a more rapid development of a chemosensory stimulus. The direct effect of endotoxin on respiratory centers also cannot be ruled out as it can induce alterations in respiratory motor output independently of fever response and this effect is partly mediated through the cyclooxygenase pathway (Preas et al., 2001). The effect of peripherally administered LPS may also be mediated by catecholaminergic and serotonergic neurotransmission in discrete brainstem nuclei (Molina-Holgado and Guaza, 1996). During fever, besides apparent thermal response, an effect of endotoxin on vagal sensory receptors of the airways and lungs must be considered. Endotoxemia is known to cause the local release of various chemical mediators in lung tissue and elicits bronchoconstriction, increased lung stiffness, and tissue edema (Plitman and Snapper, 1994). It is possible that these endotoxininduced consequences may serve as chemical and mechanical
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stimuli to activate lung vagal sensory receptors. Several investigators (Long et al., 1996; Tang et al., 1998; Lai et al., 2002) have suggested that various local or reflex pulmonary responses to endotoxin are linked to the stimulation of lung vagal sensory receptors including C-fibers. Attenuation of Hering–Breuer reflex in LPS-induced fever may represent a physiological mechanism to facilitate and to maintain thermal tachypnoea in the situation of increased body temperature. On the other hand, it also indicates affected control of breathing that can lead to respiratory instability and fatal apnea, and contribute to the pathophysiology of sudden infant death syndrome (SIDS). Multifactorial etiology of SIDS include bacterial toxemia, increase in core temperature and autonomic dysfunction (Gilbert et al., 1992; Stoltenberg et al., 1994; Voss et al., 2004) and thus decreased power of Hering–Breuer reflex in fever probably reflects altered autonomic function mediated via vagal mechanisms in control of breathing. Although a-chloralose is commonly used in rat models to investigate vagal reflexes (e.g. Aleksandrov et al., 2009) the effect of anesthesia should not be forgotten in this kind of studies (Rubini, 2011). Anesthetics might have an effect on the fever response and they also have potential to influence brain temperature and thus different physiological functions (Zhu et al., 2004). However, it can be suggested with caution that changes in neural control of breathing would occur in spontaneous pathological situations, such as fever.
5. Conclusion Altered neural control of breathing demonstrated by decreased power of Hering–Breuer inflation reflex in conditions of LPSinduced fever may play a physiological role in thermal tachypnoea as well as in the origin of respiratory instability accompanying febrile response.
Acknowledgment Authors thank D. Kuliskova, Ing. M. Petraskova, and Ing. M. Hutko for technical assistance. The study was supported by Grant of Ministry of Education VEGA no. 1/0062/08 and by the Center of Experimental and Clinical Respirology no. 26220120004, co-financed from EU sources. References Aleksandrov, V.G., Mercuriev, V.A., Ivanova, T.G., Tarasievich, A.A., Aleksandrova, N.P., 2009. Cortical control of Hering–Breuer reflexes in anesthetized rats. Eur. J. Med. Res. 14 (Suppl. 4), 1–5. Brozmanova, A., Jochem, J., Javorka, K., Zila, I., Zwirska-Korczala, K., 2006. Effects of diuretic-induced hypovolemia/isosmotic dehydration on cardiorespiratory
responses to hyperthermia and its physical treatment. Int. J. Hyperthermia 22 (2), 135–147. Gilbert, R., Rudd, P., Berry, P.J., Fleming, P.J., Hall, E., White, D.G., Oreffo, V.O., James, P., Evans, J.A., 1992. Combined effect of infection and heavy wrapping on the risk of sudden unexpected infant death. Arch. Dis. Child. 67 (2), 171–177. Haldane, J.S., 1905. The influence of high air temperatures. Am. J. Epidemiol. 55, 497–513. Javorka, K., Calkovska, A., Petraskova, M., Gecelovska, V., 1996. Cardiorespiratory parameters and respiratory reflexes in rabbits during hyperthermia. Physiol. Res. 45 (6), 439–447. Korpas, J., Tomori, Z., 1979. Cough and Other Respiratory Reflexes. Karger, Basel. Lai, C.J., Ho, C.Y., Kou, Y.R., 2002. Activation of lung vagal sensory receptors by circulatory endotoxin in rats. Life Sci. 70 (18), 2125–2138. Long, N.C., Frevert, C.W., Shore, S.A., 1996. Role of C fibres in the inflammatory response to intratracheal lipopolysaccharide. Am. J. Physiol. 271, 425–431. Merazzi, D., Mortola, J.P., 1999. Hering–Breuer reflex in conscious newborn rats: effect of changes in ambient temperature during hypoxia. J. Appl. Physiol. 87 (5), 1656–1661. Molina-Holgado, F., Guaza, C., 1996. Endotoxin administration induced differential neurochemical activation of the rat brain stem nuclei. Brain Res. Bull. 40 (3), 151–156. Morrison, S.F., Nakamura, K., 2011. Central neural pathways for thermoregulation. Front. Biosci. 16, 74–104. Plitman, J.D., Snapper, J.R., 1994. Effects of endotoxin on airway function. In: Brigham, K.L. (Ed.), Endotoxin and the Lungs. Marcel Dekker, New York, pp. 133–152. Preas 2nd, H.L., Jubran, A., Vandivier, R.W., Reda, D., Godin, P.J., Banks, S.M., Tobin, M.J., Suffredini, A.F., 2001. Effect of endotoxin on ventilation and breath variability: role of cyclooxygenase pathway. Am. J. Respir. Crit. Care Med. 164 (4), 620–626. Richards, S.A., 1968. Vagal control of thermal panting in mammals and birds. J. Physiol. 199 (1), 89–101. Robertshaw, D., 2006. Mechanisms for the control of respiratory evaporative heat loss in panting animals. J. Appl. Physiol. 101, 664–668. Rubini, A., 2011. The effect of body warming on respiratory mechanics in rats. Respir. Physiol. Neurobiol. 175, 255–260. Sebai, H., Ben-Attia, M., Sani, M., Aouani, E., Ghanem-Boughanmi, N., 2009. Protective effect of resveratrol in endotoxemia-induced acute phase response in rats. Arch. Toxicol. 83, 335–340. Siniaia, M.S., Young, D.L., Poon, C.S., 2000. Habituation and desensitization of the Hering–Breuer reflex in rat. J. Physiol. 523, 479–491. Stoltenberg, L., Sundar, T., Almaas, R., Storm, H., Rognum, T.O., Saugstad, O.D., 1994. Changes in apnea and autoresuscitation in piglets after intravenous and intrathecal interleukin-1 beta injection. J. Perinat. Med. 22 (5), 421–432. Tang, G.J., Kou, Y.R., Lin, Y.S., 1998. Peripheral neural modulation of endotoxininduced hyperventilation. Crit. Care Med. 26, 1558–1563. Trippenbach, T., 2001. Vagal afferent activity and body temperature in 3 to 10day-old and adult rats. Can. J. Physiol. Pharmacol. 79 (4), 303–309. Tryba, A.K., Ramirez, J.M., 2004. Hyperthermia modulates respiratory pacemaker bursting properties. J. Neurophysiol. 92 (5), 2844–2852. Voss, L.J., Bolton, D.P.G., Galland, B.C., Taylor, B.J., 2004. Endotoxin effects on markers of autonomic nervous system function in the piglet: implications for SIDS. Biol. Neonate 86, 39–47. Werner, M.F., Fraga, D., Melo, M.C., Souza, G.E., Zampronio, A.R., 2003. Importance of the vagus nerve for fever and neutrophil migration induced by intraperitoneal LPS injection. Inflamm. Res. 52, 291–296. White, M.D., 2006. Components and mechanisms of thermal hyperpnea. J. Appl. Physiol. 101 (2), 655–663. Zhu, M., Nehra, D., Ackerman, J.J., Yablonskiy, D.A., 2004. On the role of anesthesia on the body/brain temperature differential in rats. J. Therm. Biol. 29 (7-8), 599–603. Zila, I., Brozmanova, A., Javorka, M., Calkovska, A., Javorka, K., 2007. Effects of hypovolemia on hypercapnic ventilatory response in experimental hyperthermia. J. Physiol. Pharmacol. 58 (Suppl. 5, Pt. 2), 781–790.
Contents lists available at SciVerse ScienceDirect
Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Short Communication
Lipopolysaccharide-induced fever alters Hering–Breuer reflex in anesthetized rats I. Zila, D. Mokra, M. Javorka, K. Javorka, A. Calkovska n Department of Physiology, Jessenius Faculty of Medicine, Comenius University, Mala Hora 4, 036 01 Martin, Slovakia
a r t i c l e i n f o
abstract
Article history: Received 14 December 2011 Accepted 27 April 2012 Available online 8 May 2012
The aim of the study was to test the hypothesis that control of breathing via Hering–Breuer reflex (HB) is influenced by lipopolysaccharide (LPS)-induced fever in rats. Animals were injected intraperitoneally with LPS and control group with an equivalent volume of saline. HB reflex was elicited by inflations of the lungs followed by occlusion of the airways under control of esophageal pressure. Duration of HB reflex (Tapnoe) was continuously reduced as body temperature rose during experiment. Compared to normothermic controls, animals with fever had significant shortening of Tapnoe at 240 min and 300 min after LPS administration. Fever was further accompanied by a reduction in the strength of HB reflex (inhibitory ratio, IR). In comparison with controls, significant decrease of IR was observed at 300 min after LPS injection. Conclusion: altered neural control of breathing demonstrated by decreased power of Hering–Breuer inflation reflex in conditions of LPS-induced fever may facilitate thermal tachypnoea and/or play a role in the origin of respiratory instability accompanying febrile response. & 2012 Published by Elsevier Ltd.
Keywords: Control of breathing Hering–Breuer reflex Lipopolysaccharide Fever Rat model
1. Introduction The control in body temperature (BT) interacts with control of breathing and thus elevation in BT induces respiratory and cardiovascular changes both in humans and animals. Cardiorespiratory responses have been extensively studied in conditions of exogenous increase in core temperature (e.g. Brozmanova et al., 2006; Zila et al., 2007; Rubini, 2011), but rarely in fever. Investigation of respiratory reflexes may serve as a tool to assess neural control of breathing. Hering–Breuer (HB) reflex is elicited by activation of pulmonary slowly adapting stretch receptors and it leads to inhibition of inspiration and prolongation of expiration in order to prevent lung overinflation (Korpas and Tomori, 1979). HB reflex was considered to be a control mechanism with relatively low flexibility, however, recent studies indicate its possible modulation under specific conditions (Siniaia et al., 2000). Power of Hering–Breuer reflex is influenced mainly by chemical control of breathing and metabolic rate (Merazzi and Mortola, 1999) while both these factors are sensitive to body temperature. Moreover, vagal receptors themselves show temperature-sensitivity (Trippenbach, 2001).
n
Corresponding author. Tel.: þ421 43 2633422. E-mail addresses: [email protected] (I. Zila), [email protected] (D. Mokra), [email protected] (M. Javorka), [email protected] (K. Javorka), [email protected] (A. Calkovska). 0306-4565/$ - see front matter & 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jtherbio.2012.04.008
Taken together, increase in BT modifies breathing pattern and contributes to respiratory instability in thermal stress (Voss et al., 2004). The effects of changing vagal neural mechanisms in endotoxin-induced fever are unknown and the pathways mediating thermoregulatory influences on respiratory activity remain to be investigated (Morrison and Nakamura, 2011). Therefore, the aim of the study was to investigate the effect of fever induced by administration of lipopolysaccharide on Hering–Breuer reflex in anesthetized rats and to test the hypothesis that fever significantly affects vagally mediated ventilatory responses.
2. Materials and methods 2.1. Experimental animals and procedures 2.1.1. Animals The experiments were performed on 18 adult male rats (Wistar) with a mean body mass of 34776 g (mean 7SEM).
2.1.2. Material LPS: purified lyophilized phenol extract of Escherichia coli (026:B6, Sigma) was dissolved in sterile saline and frozen in aliquots. Before use, the LPS was diluted in sterile saline and injected intraperitoneally.
476
I. Zila et al. / Journal of Thermal Biology 37 (2012) 475–478
2.2. Design of the study The study was performed with accordance to EU Directive 2010/63/EU for animal experiments and approved by the Local Ethics Committee of the Jessenius Faculty of Medicine, Comenius University in Martin. The animals were anaesthetized with a mixture of 600 mg kg 1 urethane and 60 mg kg 1 a-chloralose intraperitoneally. They were tracheotomized and breathed spontaneously through a tracheal tube. The airflow was recorded through the Fleisch head of a pneumotachograph connected to the tracheal tube and tidal volume (VT) was obtained by electronic integration of the airflow signal (ADInstruments Pty Ltd.). The esophageal catheter was placed to record esophageal pressure during ventilation. The polyethylene catheter inserted into the femoral artery was used for arterial blood pressure monitoring and blood samples withdrawal. Blood gases ðPaO2 , PaCO2 Þ and pH analysis was performed using a blood gases analyzer (Rapidlab; Bayer Diagnostics, Germany) and values were corrected for actual body temperature. Total antioxidant status (TAS) was quantified in plasma using ABTS radical formation kinetics (Randox TAS kit, Randox Laboratories Ltd., UK). Differential white blood cell count was determined microscopically after staining by Pappenheim in peripheral blood before and 5 h after LPS/saline instillation. Colonic body temperature (Tc) was continuously measured with thermocouple (MiniLogger) inserted 4–5 cm into the anus. 2.3. Experimental protocol The animals were randomly divided into two groups. Animals of LPS group (n ¼9) received intraperitoneally LPS (100 mg kg 1) and control group (n¼9) received an equivalent volume of sterile saline. All recordings were done before (base) and 60, 120, 180, 240 and 300 min after LPS or saline administration. Hering–Breuer (HB) reflex was elicited by inflations of the lungs followed by occlusion of the airways. Inflation volume was set as 1.5x of mean tidal volume that was estimated from 10 breaths before each test. Inflations were performed at the end of inspiratory phase of breathing cycle (Fig. 1) and controlled by changes in esophageal pressure. Occlusion was maintained until first inspiratory effort occurred as indicated by changes in esophageal pressure. The duration of Hering–Breuer reflex (Tapnoe) was determined as the total time from the beginning of the expiration immediately before the lung inflation to the onset of the next inspiration during the maintained inflation. The strength of Hering–Breuer reflex was quantified as inhibitory ratio (IR) and was calculated as the ratio between Tapnoe and average expiratory time of five breaths preceding the lung inflation (according to Merazzi and Mortola (1999)). 2.4. Statistical analysis The statistical software package SYSTAT 6.0.1 (SPSS Inc., 1996) was used for data analysis. Data were tested for normality of
distribution using Kolmogorov–Smirnov test. Between-group differences were analyzed using Mann–Whitney test, and withingroup differences with Wilcoxon test. Data are expressed as means7S.E.M., differences are considered significant when Po0.05.
3. Results Administration of lipopolysaccharide was accompanied by continuous rise in colonic body temperature compared to salinetreated controls. The intraperitoneal injection of 100 mg kg 1 induced monophasic thermogenic reaction in all animals with peak registered 300 min after LPS administration (Fig. 2). There were no differences in initial values of neutrophil count in the peripheral blood in control vs. LPS groups. Administration of endotoxin evoked systemic inflammatory response accompanied by an increase in neutrophil count at the end of the experiments: 73.3710.8% in LPS group vs. 50.4719.1% in controls; Po0.01. Total antioxidant status was significantly lower in LPS-treated group in comparison with controls (0.86270.082 mmol/l vs.1.0097 0.15 mmol/l; Po0.05). Data on duration and strength of Hering–Breuer reflex are summarized in Figs. 3 and 4. The duration of HB reflex was continuously reduced as body temperature rose during experiment. In comparison with normothermic controls, LPS animals exhibited significant shortening of Tapnoe at 240 min after LPS administration—Tapnoe240: 2.270.3 s vs. 3.470.3 s, Po0.05, as well as at the maximum temperature of 38.6 1C at 300 min after endotoxin injection—Tapnoe300: 1.670.2 s vs. 3.570.3 s, P o0.01 (Fig. 3). Progression of fever response was accompanied by the reduction in the strength of Hering–Breuer reflex (Fig. 4). Inhibitory ratio (IR) was reduced at 180, 240 and 300 min after lipopolysaccharide administration compared to baseline. In comparison with controls, significant decrease of IR was observed at 300 min from LPS injection: 5.2 70.2 vs. 9.2 70.3; Po0.01. The initial values of respiratory parameters did not differ between the groups. In controls VT before and after experiment was unchanged (1.9 70.3 mL vs. 1.870.2 mL; P40.05 ) as was the respiratory rate (97711 breaths per minute; bpm vs. 103714 bpm; P40.05). In LPS group, minute ventilation progressively increased with elevation of body temperature as a result of significant increase in respiratory rate from baseline 105711 bpm to 258719 bpm (Po0.01) at 300 min after LPS administration. Tidal volume in LPS animals was reduced to 1.270.2 mL (at Tc300) in comparison with baseline value (1.870.3 mL, Po0.05). No significant differences were present in absolute values of PaO2 , PaCO2 and pH between LPS and control groups or in values compared to baseline. At 300 min after LPS administration PaCO2 tended to decrease but the drop was not significant (data not shown).
Fig. 1. Record of airflow during inflation of the lungs in a single experiment. Te—duration of expiration, Tapnoe—duration of Hering–Breuer reflex, V —airflow, sec—second.
I. Zila et al. / Journal of Thermal Biology 37 (2012) 475–478
Fig. 2. Colonic body temperature (oC) measured before LPS/saline administration (Tcbase) and 60, 120, 180, 240 and 300 min after LPS/saline administration P o0.05.
Fig. 3. Duration (Tapnoe) of HB reflex measured before LPS/saline administration (Tcbase) and 60, 120, 180, 240 and 300 min after LPS/saline administration. Values are expressed as mean 7SEM. Within-group comparison to baseline (before LPS/ saline injection), # Po 0.05; between-group comparison, *P o 0.05, **P o 0.01.
Fig. 4. Strength (inhibitory ratio, IR) of HB reflex measured before LPS/saline administration (Tcbase) and 60, 120, 180, 240 and 300 min after LPS/saline administration. Values are expressed as mean 7 SEM. Within-group comparison to baseline (before LPS/saline injection), # Po 0.05; between-group comparison, **P o 0.01.
4. Discussion Lipopolysaccharide (LPS) derived from the cell wall of Gramnegative bacteria can evoke fever and systemic inflammatory response. Fever is thought to provide an optimal hyperthermic environment for mounting host defense against invading bacteria and viruses (Morrison and Nakamura, 2011). In this study, thermogenic response in LPS-injected animals was accompanied by increase in neutrophil count in peripheral
477
blood and by reduction in total antioxidant status as a consequence of production of wide spectrum of inflammatory mediators and reactive oxygen species (ROS) (Werner et al., 2003; Sebai et al., 2009). The body temperature-induced changes in breathing pattern are evoked by alterations in the peripheral temperature, or modified by shifts in the central body temperature, eventually by other mechanisms. Hyperpnoea associated with elevated core temperature was described for the first time by Haldane (1905) and since then numerous studies have been focused on investigation of breathing control in elevated-body temperature conditions. In some species, a rise in body temperature develops a specialized breathing pattern known as thermal tachypnoea that is mediated by the thermoregulatory system in the preoptic area of the hypothalamus. Thermal tachypnoea can, in some mammals and birds, be controlled by central mechanisms alone, while in others it depends on extrinsic stimuli mediated by way of the vagus nerve (Richards, 1968). During this pattern of ventilation, with an elevated functional residual capacity, a high frequency of breathing and a reduced tidal volume, the upper airways are preferentially ventilated and therefore blood gases and pH are maintained in a physiological range (White, 2006). In our experiments, increase in BT in rats with fever was characterized by rapid shallow breathing with significant increase in respiratory rate and decrease in tidal volumes typical for thermal tachypnoea. PaCO2 in LPS animals tended to decrease suggesting increased ventilation that is largely confined to the dead space with only a small increase in alveolar ventilation (Robertshaw, 2006). In these conditions HB inflation reflex was reduced in its duration and strength. Although mechanisms that elicit a rise in body temperature in exogenous increase in core temperature and fever are different, the results are in accordance with previous study of our group in hyperthermic rabbits (Javorka et al., 1996). However, temperature can also directly affect the activity of the respiratory neural network located in the medulla. For ¨ instance, area of pre-Botzinger complex was suggested to play critical role in a change of breathing pattern during thermal stress (Tryba and Ramirez, 2004). The magnitude of the reflex inhibition of ventilation during sustained inflation is probably determined at different levels within the loop Hering–Breuer reflex. Slowly adapting stretch receptors, chemoreceptors and the central effectiveness and processing of vagal inputs seem to be most important as all these components are sensitive to changes in temperature (Merazzi and Mortola, 1999; Zila et al., 2007) and thus an altered sensitivity could play a key role in modulation of vagally mediated ventilatory responses. Particularly, interaction between chemosensitivity and temperature is suggested to have stronger influence on breathing than the thermogenic inhibition of vagal ventilatory mechanisms and the change in HB reflex in febrile rats could result from a more rapid development of a chemosensory stimulus. The direct effect of endotoxin on respiratory centers also cannot be ruled out as it can induce alterations in respiratory motor output independently of fever response and this effect is partly mediated through the cyclooxygenase pathway (Preas et al., 2001). The effect of peripherally administered LPS may also be mediated by catecholaminergic and serotonergic neurotransmission in discrete brainstem nuclei (Molina-Holgado and Guaza, 1996). During fever, besides apparent thermal response, an effect of endotoxin on vagal sensory receptors of the airways and lungs must be considered. Endotoxemia is known to cause the local release of various chemical mediators in lung tissue and elicits bronchoconstriction, increased lung stiffness, and tissue edema (Plitman and Snapper, 1994). It is possible that these endotoxininduced consequences may serve as chemical and mechanical
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stimuli to activate lung vagal sensory receptors. Several investigators (Long et al., 1996; Tang et al., 1998; Lai et al., 2002) have suggested that various local or reflex pulmonary responses to endotoxin are linked to the stimulation of lung vagal sensory receptors including C-fibers. Attenuation of Hering–Breuer reflex in LPS-induced fever may represent a physiological mechanism to facilitate and to maintain thermal tachypnoea in the situation of increased body temperature. On the other hand, it also indicates affected control of breathing that can lead to respiratory instability and fatal apnea, and contribute to the pathophysiology of sudden infant death syndrome (SIDS). Multifactorial etiology of SIDS include bacterial toxemia, increase in core temperature and autonomic dysfunction (Gilbert et al., 1992; Stoltenberg et al., 1994; Voss et al., 2004) and thus decreased power of Hering–Breuer reflex in fever probably reflects altered autonomic function mediated via vagal mechanisms in control of breathing. Although a-chloralose is commonly used in rat models to investigate vagal reflexes (e.g. Aleksandrov et al., 2009) the effect of anesthesia should not be forgotten in this kind of studies (Rubini, 2011). Anesthetics might have an effect on the fever response and they also have potential to influence brain temperature and thus different physiological functions (Zhu et al., 2004). However, it can be suggested with caution that changes in neural control of breathing would occur in spontaneous pathological situations, such as fever.
5. Conclusion Altered neural control of breathing demonstrated by decreased power of Hering–Breuer inflation reflex in conditions of LPSinduced fever may play a physiological role in thermal tachypnoea as well as in the origin of respiratory instability accompanying febrile response.
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