Effect of bilateral vagotomy on oxygenation, and breathing movements in fetal sheep SHABIH U. HASAN AND ANITA RIGAUX Department of Paediatrics and Reproductive Medicine Calgary, Alberta T2N 4N1, Canada HASAN, SHABIH U., AND ANITA RIGAUX. Effect of bilateral vagotomy on oxygenation, arousal, and breathing movements in fetal sheep. J. Appl. Physiol. 73(4): 1402-1412,1992.-To investigate the effects of bilateral cervical vagotomy on arousal and breathing responses, we studied eight sham-operated and eight chronically instrumented unanesthetized vagotomized sheep fetuses between 136 and 144 days of gestation (term ~147 days). Each fetus was instrumented to record sleep states, diaphragmatic electromyogram, blood pressure, pH, and blood gas tensions. In a randomized order, fetal lungs were distended with four different 0, concentrations: 0 (100% N2), 21, 50, and 100% at a continuous positive airway pressure of 30 cmH,O via an in situ Y-endotracheal tube. Under control conditions, inspiratory time and the duration of the single longest breathing episode decreased from 598 * 99 (SD) ms and 24 * 10 min in sham group to 393 * 162 ms and 11.0 t 3.0 min in vagotomized group (P = 0.04 and O.O33), respectively. In response to lung distension with 100% N,, breathing time decreased from 44 2 17 to 20 t 18% (P = 0.045) in sham-operated fetuses, whereas it remained unchanged in the vagotomized group. In response to 100% O,, fetal arterial PO, increased in five of eight fetuses sham-operated from 18.2 Ifr 5.1 to 227 t 45 Torr (P < 0.0001) and in six of eight vagotomized fetuses from 18.5 t 4.4 to 172 t 39 Torr (P < 0.001). Although arousal was observed in all oxygenated fetuses at the onset of breathing, the duration of arousal was markedly attenuated in vagotomized fetuses (14 * 10 vs. 46 t 29 min in sham group; P = 0.024). Frequency and amplitude of breathing and respiratory output (frequency X amplitude) increased only in sham group (P = 0.02,0.004, and 0.0002, respectively). We conclude that in response to lung distension and oxygenation, arousal and stimulation of breathing during active and quiet sleep are critically dependent on intact vagal nerves. lung distension; vagal afferents; control of breathing; arousal; active sleep; quiet sleep; fetal breathing
fetal
BREATHING is intermittent in utero as opposed to continuous after birth. In sheep, during late gestation, episodic fetal breathing movements (FBM) occur for 40% of the total time and normally only during low-voltage electrocortical activity (LV-ECoG) (17). The control mechanisms for the episodic FBM or the establishment of continuous breathing at birth remain unknown. Recent studies have shown that an increase in fetal arterial PO, (Pa& may induce continuous FBM (3). In our previous study, initiation of FBM in response to an increase in fetal Paoz was always associated with arousal (27). Furthermore, both arousal and stimulation of breathing during high-voltage (HV)-ECoG were found to be critically 1402
0161-7567/92 $2.00 Copyright
0
Research
arousal,
Group, The University
of Calgary,
dependent on fetal maturity, almost always occurring after 135 days of gestation (term = 147 t 2 days) (27). It has recently been shown that vagal afferents have a significant role in breathing pattern as well as in ventilation during the newborn period, which is in contrast with their role in adults (20). Furthermore, experiments by Johnson (34) showed that in ll- to 43-day-old lambs, a sustained distension of lungs at lo-20 cmH,O pressure produced an initial decrease, which is followed by an increase in respiratory frequency above control levels (34). Other studies have shown that the central respiratory center output is more dependent on afferent information provided by the vagus in the very young infant than in the older child or adult and that this vagal input was more important during quiet sleep (21, 52). Because the increase in fetal PaoZ in our studies was achieved by continuous positive airway pressure (CPAP) and 100% O,, we could not rule out an interaction between oxygenation plus lung distension and mechanoreceptor feedback in the initiation/maintenance of continuous breathing and we designed a study to test the hypothesis that bilateral vagotomy would either prevent or attenuate continuous FBM induced by lung distension and oxygenation. METHODS Surgical preparation. Sixteen time-dated pregnant ewes of mixed breed underwent surgery between 129 and 133 days of gestation. Experiments were done between 136 and 144 days of gestation (term = 147 t 2 days). Although experiments were not done until 136 days of gestation, surgery at an early gestational age was done to reduce the risk of labor. The details of surgical preparation have been given elsewhere (27). Briefly, with the animals under general anesthesia, using 4% halothane for induction and 1.5-2% for maintenance, the abdominal and uterine walls of the ewe were opened through a midline incision and the fetus was partially exteriorized. The instrumentation of the fetus included implantation of electrodes, insertion of carotid arterial and jugular venous catheters, and placement of an endotracheal tube. Four bipolar Teflon-coated stainless steel electrode wires (Cooner, Chatsworth, CA) were implanted to record 1) ECoG, 2) electrooculogram (EOG), 3) nuchal electromyogram (EMGNK), and 4) diaphragm EMG (EMGdi). To record the ECoG, electrodes were implanted bilaterally over the parietal area using rubber pads and cyanoacrylate glue, whereas EOG was obtained
1992 the American
Physiological
Society
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by implanting a silver-coated oval-shaped electrode in the lateral rectus muscles of one eye. A pair of electrodes was sutured into the lateral neck muscles, 2.5 cm apart and 5 cm below the occiput to record EMGNK. EMGdi was obtained by implanting the electrode wires in the right hemidiaphragm through an incision in the right 10th intercostal space. ECoG, EOG, and EMGNK were used to define fetal behavioral states. A 5 to 7-cm midline ventral incision, starting 5 mm below the thyroid cartilage, was made to expose the jugular veins, carotid arteries, vagal nerves, and trachea. Polyvinyl catheters (1.0 mm ID and 2.0 mm OD; Portex, Hythe, Kent, UK) were placed in the fetal carotid artery and jugular veins to withdraw arterial blood samples to measure pH and blood gas tensions and to infuse fluids and antibiotics, respectively. To apply CPAP to the lung, fetal trachea was cannulated with a Y-shaped endotracheal tube (4.8 mm ID and 7.9 mm OD; Tygon, Norton Plastics, Akron, OH). The endotracheal tube was advanced for 5-6 cm and transfixed (0 silk) to prevent its dislodgement from the trachea. In addition, two sutures (4-silk) were placed around the trachea to prevent air leaks during the lung distension experiments. The proximal free ends of the Y-endotracheal tubes were attached to two polypropylene tubes (4.8 mm ID, 7.8 mm OD; Tygon) that served as inspiratory and expiratory tubes. Eight fetuses underwent bilateral vagotomy while the remaining eight were sham operated. The vagus nerves were carefully separated from the carotid artery in the cervical region by blunt dissection, and 2.5- to 3.0-cmlong pieces were removed from both vagi. Because our preliminary results from vagotomized fetuses showed attenuation of FBM and it was not clear whether the attenuation of FBM was due to denervation of aortic chemoreceptors or other vagal afferents, the sham surgery included identification but not section of vagal as well as carotid sinus nerves. According to a modified technique originally described by Itskovitz and Rudolph (31), through a midline incision the superior laryngeal artery and nerves were identified and left intact. Through further blunt dissection, superior cervical ganglion and glossopharyngeal nerves were identified. The carotid sinus nerve arises from the glossopharyngeal nerve and courses from the medial aspect of the hyoid bone to the carotid body and sinus area, which are situated at the bifurcation of carotid and occipital arteries. Similar procedure was repeated on the contralateral side and the incisions were sewn in layers. This procedure added 15 min extra on each side to surgery time. Once the surgical procedures and instrumentation were complete, two amniotic catheters were sutured to the back of the fetal head, and the fetus was returned to the uterus and all maternal incisions were sewn in layers. One of the amniotic catheters (polyvinyl, 2.0 mm ID and 3.0 mm OD; Portex) was used as a reference zero value for fetal arterial blood pressure and also to monitor the amniotic pressure. The second amniotic catheter (polypropylene, 4.8 mm ID, 7.9 mm OD; Tygon) was connected to one of the two extensions of the Y-endotracheal tube to facilitate tracheoamniotic fluid flow, whereas the second extension of the endotracheal tube remained plugged when no experiments were being done.
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A polyvinyl catheter (2.0 mm ID, 3.0 mm OD; Portex) was placed in the jugular vein of the ewe to administer fluids and antibiotics. The fetal electrode wires, vascular catheters, and endotracheal tube extensions were exteriorized through the left maternal flank and stored in a cloth pouch sewn adjacent to the exteriorized site. Postoperatively, the ewes received 1 g of cephalothin (Keflin, Eli Lilly Canada, Toronto, Ontario) and 80 mg of gentamicin (Cidomycin, Roussel Canada, Montreal, Quebec) intravenously and 20 mg of gentamicin into the amniotic cavity, whereas the fetuses received 150 mg of cephalothin and 10 mg of gentamicin intravenously twice daily for 5 days. Also, fetal and maternal vascular catheters were flushed twice daily with heparinized isotonic saline (10 U heparin/ml). Data acquisition and analysis. The amniotic pressure and fetal arterial blood pressure were calibrated twice daily and recorded with Statham P23 ID pressure transducers (Gould Instruments Division, Cleveland, OH). Electrophysiological signals from ECoG, EOG, EMGNK, and EMGdi were appropriately amplified and filtered (NeuroLog IOOAK, l02G, and 125/6; Medical Systems, Greenvale, NY) and, along with the amniotic pressure and blood pressure, were recorded on an eightchannel chart recorder (Gould 2800s; Gould). In addition, all signals were digitized (Neurocorder DR-886; Neurodata Instruments, New York, NY) and stored on a videocassette recorder (model FVH-C4000, Fisher). Respiratory and behavioral patterns during control and experimental conditions included manual measurements of 1) percentage of breathing time during total time (the fetus was considered to be breathing when there were at least six breaths/min, and measurements of control percentage of breathing time included equal numbers of LVand HV-ECoG periods); 2) total duration of LV-ECoG and HV-ECoG as a percentage of total time; 3) percentage of time breathing in LV-ECoG (percentage of breathing time during LV-ECoG/total LV-ECoG time); 4) percentage of time breathing in HV-ECoG (percentage of time breathing during HV-ECoG/total HV-ECoG time; both percentage of time breathing in LV-ECoG and percentage of time breathing during HV-ECoG are the most precise variables indicating the relationship between breathing and electrocortical activity); 5) single longest breathing episode in minutes (single longest breathing episode was a breathing episode where breathing was not interrupted by an apneic period); and 6) time lag to the onset of continuous breathing movements if present. Continuous breathing episodes were defined as the breathing episodes that not only remained continuous during both LV- and HV-ECoG but also terminated only when the experimental condition was changed. The diaphragm EMG signal was integrated and from the integrated signal frequency (f), number of diaphragmatic contractions per minute of breathing time, amplitude of breathing (JEMGd i ) in arbitrary units from the total area of the integrated diaphragmatic EMGdi, respiratory output ( EMGdi X f), and inspiratory time were measured. Ir hese analyses were done during arousal and rapid-eye-movement (REM) sleep in both sham-operated and vagotomized fetuses. Cardiovascular responses included measurements of systolic, diastolic, and mean
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1404 ECoG
VAGOTOMY
I
I I
I
I
I I
CONTROL
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1
I
I ‘t Tracheal
Suction
CPAP 3’0 cm H,O plus Randomized O2 % 0% (100% Nitrogen) 21% 50% 100% FIG. 1. Schematic representation of experimental design. First, control period consisting of breathing and nonbreathing episodes was obtained. Tracheal suction to remove tracheal fluid was followed by fetal lung distension with 4 different concentrations of 0, [0 (100% Nz), 21,50, and lOO%] at continuous positive airway pressure (CPAP) of 30 cmH,O in a randomized order. All changes were made during high-voltage electrocortical (ECoG) activity.
blood pressure and heart rate. Blood pressure was recorded continuously during control and experiments. Both blood pressure and heart rate were measured every 10 min in both sham-operated and vagotomized fetuses. The frequency response of our pressure recording system was confirmed with a Millar multifunction pressure generator (waveform generator, model WGA-200; Millar Instruments, Houston, TX) and was found to be adequate. Electrographic criteria were used to define active sleep, quiet sleep, and awake states (29, 43, 51). Active sleep was defined by the simultaneous presence of LVECoG and eye movements and absence of neck muscle tone, whereas quiet sleep was defined by the simultaneous presence of HV-ECoG and neck muscle tone and absence of eye movements. Fetal wakefulness was defined by the simultaneous presence of LV-ECoG, eye movements, and neck muscle tone. Experimental protocol. No experiments were done for at least 4 days after surgery and only after fetal physiological variables were within normal range. The normal ranges for physiological variables were the pH >7.30, arterial PCO, (PacoJ 18 Torr. The breathing time was considered normal when it was >25% of the total time. We did not record any control periods or perform any experiments during labor. The experimental design is shown in Fig. 1. The physiological variables, amniotic pressure, blood pressure, ECoG, EOG, EMGNK, and EMGdi were displayed on the chart recorder at a speed of 0.05 mm/s overnight and at variable speeds (0.05-10 mm/s) during the experiments. In addition, all control and experiments were recorded on a videocassette for future analyses of all recorded variables. Experiments were not started until 1000 h to avoid the morning trough period of FBM. Once the control period of at least 3 h was obtained, fetal trachea was suctioned through one of the endotracheal loops, the patency of both loops was checked, and fetal lungs were distended at a CPAP of 30 cmH,O, relative to atmospheric pressure, with four different concentrations of 0, [0 (100% N2), 21, 50, and lOO%] in a randomized order (Baby Bird Ventilator; Bird, Palm Springs, CA). Randomization was achieved by drawing without replacement one of the four concealed 3-in. X 3-in. pieces of papers bearing the gas concentration. This procedure was performed before each experiment. N, was added to
AND
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the protocol as a control for gaseous distension. The variables obtained during experiments were not compared with the overnight controls but with the controls obtained between 1000 and 1300 h on a given day. All gas mixtures were humidified and warmed to 39.5”C before administration (model 328; Fisher Paykel, Allied Products Medical Division, 25 Panmure, Auckland, New Zealand). 0, concentration was adjusted by the Baby Bird 0, blender as well as by an 0, analyzer (model 5584EC; Hudson-Ventronics Division, Temecula, CA). Each 0, concentration was maintained for -2 h, and the change in gas mixture was done only during HV-ECoG to avoid the confounding effects of fetal breathing that occur during LV-ECoG. If the fetuses continued to breathe during HV-ECoG, we waited for 1 h. However, if the fetus continued to breathe beyond that period, the change in gas concentration was made during HV-ECoG according to the randomized sequence. This step might have underestimated the duration of the single longest breathing episodes, especially among the sham-operated fetuses, but was taken to maintain consistency during the experiments. Arterial pH and blood gas tensions were analyzed during control period and during lung distension with various gas mixtures. These analyses were done during both LV- and HV-ECoG. The blood samples were kept on ice until the time of analysis, and the temperature of the blood gas analyzer was modified to 39.5OC (ILl301; Instrumentation Laboratory System, Lexington, MA). Statistical analysis. All recorded physiological variables in sham-operated and vagotomized fetuses were compared within each group as well as between the two groups. The effects of 0 (100% NJ, 21, 50, and 100% 0, on various variables were compared with the control values within each group, vagotomized and sham-operated, using analysis of variance for repeated measures. The significance of differences was assessed by Dunnett’s test. Further analysis of variance was done to investigate whether there were any differences between the two groups and whether those differences were dependent on the 0, concentration. Nonpaired t test was then done to detail these interactions between the vagotomized and sham-operated groups. Inspiratory times were compared by two-way analysis of variance. Characteristics of the fetuses (Table 1), with the exception of gestational age, were compared using Fisher’s exact test, whereas non1. Characteristics of sham-operated and vagotomized fetuses TABLE
Sham
Total no. Gestational age, days Number of oxygenated fetuses Wakefulness observed Breathing during LVand HV-ECoG
Operated
8 139+3
Vagotomized
P
8 138+2
0.652*
5
6
1.00-f
5
6
1.00-f.
5
0
0.025-f Q
Gestational age values are means + SD. LV-ECoG, low-voltage electrocortical activity; HV-ECoG, high-voltage ECoG. * Nonpaired t test; t Fisher’s exact test; $ significant difference between awake fetuses and fetuses that breathed during both LV- and HV-ECoG.
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VAGOTOMY
ATTENUATES
AROUSAL
100 s w I F g
I 2 Is!
50
m
0 6 .-
E
80
B
O-
o SHAM-OPERATED
a-
. VAGOTOMIZED
*t
$3 i2 a 40 IL z 0 0
z(r 3n
0 C
N2
21
50
100
2. Breathing time (A) and duration of arousal (B) in shamoperated and vagotomized fetuses under control conditions and during lung distension with 100% N, and 21,50, and 100% 0, at CPAP of 30 cmH,O. Breathing time decreased during lung distension with NS, whereas both breathing time and duration of arousal significantly increased during lung distension with 100% 0, in sham-operated group only. Changes in breathing time and duration of arousal were also significantly different from those observed in vagotomized fetuses. *P < 0.05 from control values within group; tP < 0.05 between sham-operated and vagotomized fetuses. FIG.
AND
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mized fetuses (Fig. 6). Similarly, plasma bicarbonate was 23.8 t 1.3 and 24.1 t 0.8 mM in sham-operated and vagotomized groups, whereas the base deficit was -0.9 t 1.2 and -0.7 t 1.4 mM, respectively. Although lung distension with 50% 0, in sham-operated group resulted in a small increase in Pa,, (35.0 t 16.4 Torr), it did not reach statistical significance. However, in response to 100% O,, pH decreased (P < 0.0001 and 0.036) and Paoz increased (P < 0.0001 and lOO% in blood pressure and heart rate variability among the denervated fetuses. Koos and Sameshima (35) have also reported an increased although markedly less pronounced variability in arterial blood pressure in sinoaortic-denervated fetuses. Besides the possible effects of vagotomy on pulmonary afferents as discussed above, vagal nerves also carry reflexes from chemo- and/or baroreceptors, abdominal expiratory muscles, the cardiovascular system, and abdominal viscera. Interruption of chemo- and/or baroreceptor activity can result in decreased fetal breathing movements and can also lead to respiratory failure in the newborn (11, 19, 35, 40). Therefore, this possibility needs to be investigated in our model. Bishop (6) and Bishop and Bachofen (7) showed that positive airway pressure could lead to stimulation of abdominal muscles not only during expiration but also to some extent during inspiration. Similarly, Gilmartin et al. (24) have recently shown the recruitment of various abdominal muscles during stepwise increments of positive end-expiratory pressures and vagotomy resulted in attenuation of such muscle activity. Therefore, augmentation of breathing in our studies could have been partly due to the recruitment of abdominal expiratory muscles during application of distending pressure, and such augmentation was abolished by bilateral vagotomy. In summary, we have shown that arousal and stimulation of breathing in response to lung distension and oxygenation in near-term fetal sheep can be attenuated by bilateral cervical vagotomy. Further studies involving the blood flow measurements, expiratory muscle activity, differential vagal blockade, and single-fiber recordings are warranted to dissect the mechanism(s) underlying these responses. The authors are grateful to Michelle Cavanaugh for secretarial assistance, Drs. Tak Fung and Gordon Fick for statistical analysis, and Dr. John Remmers for constructive criticism. We also acknowledge the generous financial support of the Alberta Children’s Hospital Foundation and the Alberta Lung Association. This work was presented in part at the Federation of American Societies for Experimental Biology Meeting in Washington, DC in April 1990. Address for reprint requests: S. U. Hasan, Dept. of Paediatrics, The University of Calgary, Faculty of Medicine, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Received
28 August
1991; accepted
in final
form
13 May
1992.
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28. HENDERSON-SMART, D. J., P. JOHNSON, AND M. E. MCCLELLAND. Asynchronous activity of the diaphragm during breathing in lambs. J. Physiol. Lond. 296: 22-23, 1979. 29. IOFFE, S., A. H. JANSEN, B. H. RUSSELL, AND V. CHERNICK. Sleep, wakefulness and the monosynaptic reflex in fetal and newborn lambs. Pfluegers Arch. 388: 149-157, 1980. 30. ITSKOVITZ, J., E. F. LAGAMMA, AND A. M. RUDOLPH. Baroreflex control of the circulation in chronically instrumented fetal lambs. Circ. Res. 52: 589-596, 1983. 31. ITSKOVITZ, J., AND A. M. RUDOLPH. Denervation of arterial chemoreceptors and baroreceptors in fetal lambs in utero. Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H916-H920, 1982. 32. IWAMOTO, H. S., D. TEITEL, AND A. M. RUDOLPH. Effects of birthrelated events on blood flow distribution. Pediatr. Res. 22: 634-640, 1987. 33. JANSEN, A. H., S. IOFFE, B. J. RUSSELL, AND V. CHERNICK. Influence of sleep state on the response to hypercapnia in fetal lambs. Respir. Physiol. 48: 125-142, 1982. 34. JOHNSON, P. Comparative aspects of the control of breathing during development. Wenner-Gren Cent. Int. Symp. Ser. 32: 337-352, 1980. 35. KOOS, B. J., AND H. SAMESHIMA. Effects of hypoxaemia and hypercapnia on breathing movements and sleep state in sinoaortic-denervated fetal sheep. J. Deu. Physiol. Oxf. 10: 131-144, 1988. 36. MALONEY, J. E., T. M. ADAMSON, V. BRODECKY, M. H. DOWLING, AND B. C. RITCHIE. Modification of respiratory center output in the unanesthetized fetal sheep in utero. J. Appl. Physiol. 39: 552558, 1975. 37. MARSLAND, D. W., B. J. CALLAHAN, AND D. C. SHANNON. The afferent vagus and regulation of breathing in response to inhaled COZ in awake newborn lambs. Biol. Neonate 27: 102-107, 1975. 38. MORTOLA, J. P., J. T. FISHER, AND G. SANT’AMBROGIO. Vagal control of the breathing pattern and respiratory mechanics in the adult and newborn rabbit. Pfluegers Arch. 401: 281-286, 1984. 39. MOSS, I. R., AND E. M. SCARPELLI. Generation and regulation of breathing in utero: fetal CO, response test. J. Appl. Physiol. 47: 527-531, 1979. 40. MURAI, D. T., C. H. LEE, L. D. WALLEN, AND J. A. KITTERMAN. Denervation of peripheral chemoreceptors decreases breathing movements in fetal sheep. J. Appl. Physiol. 59: 575-579, 1985. 41. PACK, A. I. Sensory inputs to the medulla. Annu. Rev. Physiol. 43: 73-90, 1981. 42. PHILLIPSON, E. A., AND G. BOWES. Control of breathing during sleep. In: Handbook of Physiology. The Respiratory System. Control of Breathing. Bethesda, MD: Am. Physiol. Sot., 1986, sect. 3, vol. II, pt. 2, chapt. 19, p. 649-690. 43. PHILLIPSON, E. A., N. H. FISHMAN, R. F. HICKEY, AND J. A. NADEL. Effect of differential vagal blockade on ventilatory response to CO, in awake dogs. J. Appl. Physiol. 34: 759-763, 1973. 44. PHILLIPSON, E. A., E. MURPHY, AND L. F. KOZAR. Regulation of respiration in sleeping dogs. J. Appl. Physiol. 40: 688-693, 1976. 45. RICHTER, D. W. Generation and maintenance of the respiratory rhythm. J. Exp. Biol. 100: 93-107, 1982. 46. RIGATTO, H., S. U. HASAN, A. JANSEN, D. GIBSON, B. NOWACZYK, AND D. CATES. The effect of total peripheral chemodenervation on fetal breathing and on the establishment of breathing at birth. In: Fetal and Neonatal Development, edited by C. T. Jones. Ithaca, New York: Perinatology, 1988, p. 613-621. 47. RIGATTO, H., D. LEE, M. DAVI, M. MOORE, E. RIGATTO, AND D. CATES. Effect of increased arterial CO2 on fetal breathing and behavior in sheep. J. Appl. Physiol. 64: 982-987, 1988. 48. SCHWIELER, G. H. Respiratory regulation during postnatal development in cats and rabbits and some of its morphological substrate. Acta Physiol. Stand. Suppl. 304: 1-123, 1968. 49. SHELDON, M. I., AND J. F. GREEN. Evidence for pulmonary-CO2 chemosensitivity: effects on ventilation. J. Appl. Physiol. 52: 11921197, 1982. 50. SULLIVAN, C. E., L. F. KOZAR, E. MURPHY, AND E. A. PHILLIPSON. Arousal, ventilatory, and airway responses to bronchopulmonary stimulation in sleeping dogs. J. Appl. Physiol. 47: 17-25, 1979. 51. SZETO, H. H., AND D. J. HINMAN. Prenatal development of sleepwake patterns in sheep. Sleep 8: 347-355, 1985. 52. THACH, B. T., I. D. FRANTZ, S. M. ADLER, H. W. TAEUSCH, AND M. E. AVERY. Vagal influence on inspiratory duration in premature infants as a function of postnatal and gestational age (Abstract). Federation Proc. 34: 358, 1975.
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fetal
BREATHING is intermittent in utero as opposed to continuous after birth. In sheep, during late gestation, episodic fetal breathing movements (FBM) occur for 40% of the total time and normally only during low-voltage electrocortical activity (LV-ECoG) (17). The control mechanisms for the episodic FBM or the establishment of continuous breathing at birth remain unknown. Recent studies have shown that an increase in fetal arterial PO, (Pa& may induce continuous FBM (3). In our previous study, initiation of FBM in response to an increase in fetal Paoz was always associated with arousal (27). Furthermore, both arousal and stimulation of breathing during high-voltage (HV)-ECoG were found to be critically 1402
0161-7567/92 $2.00 Copyright
0
Research
arousal,
Group, The University
of Calgary,
dependent on fetal maturity, almost always occurring after 135 days of gestation (term = 147 t 2 days) (27). It has recently been shown that vagal afferents have a significant role in breathing pattern as well as in ventilation during the newborn period, which is in contrast with their role in adults (20). Furthermore, experiments by Johnson (34) showed that in ll- to 43-day-old lambs, a sustained distension of lungs at lo-20 cmH,O pressure produced an initial decrease, which is followed by an increase in respiratory frequency above control levels (34). Other studies have shown that the central respiratory center output is more dependent on afferent information provided by the vagus in the very young infant than in the older child or adult and that this vagal input was more important during quiet sleep (21, 52). Because the increase in fetal PaoZ in our studies was achieved by continuous positive airway pressure (CPAP) and 100% O,, we could not rule out an interaction between oxygenation plus lung distension and mechanoreceptor feedback in the initiation/maintenance of continuous breathing and we designed a study to test the hypothesis that bilateral vagotomy would either prevent or attenuate continuous FBM induced by lung distension and oxygenation. METHODS Surgical preparation. Sixteen time-dated pregnant ewes of mixed breed underwent surgery between 129 and 133 days of gestation. Experiments were done between 136 and 144 days of gestation (term = 147 t 2 days). Although experiments were not done until 136 days of gestation, surgery at an early gestational age was done to reduce the risk of labor. The details of surgical preparation have been given elsewhere (27). Briefly, with the animals under general anesthesia, using 4% halothane for induction and 1.5-2% for maintenance, the abdominal and uterine walls of the ewe were opened through a midline incision and the fetus was partially exteriorized. The instrumentation of the fetus included implantation of electrodes, insertion of carotid arterial and jugular venous catheters, and placement of an endotracheal tube. Four bipolar Teflon-coated stainless steel electrode wires (Cooner, Chatsworth, CA) were implanted to record 1) ECoG, 2) electrooculogram (EOG), 3) nuchal electromyogram (EMGNK), and 4) diaphragm EMG (EMGdi). To record the ECoG, electrodes were implanted bilaterally over the parietal area using rubber pads and cyanoacrylate glue, whereas EOG was obtained
1992 the American
Physiological
Society
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VAGOTOMY
ATTENUATES
AROUSAL
by implanting a silver-coated oval-shaped electrode in the lateral rectus muscles of one eye. A pair of electrodes was sutured into the lateral neck muscles, 2.5 cm apart and 5 cm below the occiput to record EMGNK. EMGdi was obtained by implanting the electrode wires in the right hemidiaphragm through an incision in the right 10th intercostal space. ECoG, EOG, and EMGNK were used to define fetal behavioral states. A 5 to 7-cm midline ventral incision, starting 5 mm below the thyroid cartilage, was made to expose the jugular veins, carotid arteries, vagal nerves, and trachea. Polyvinyl catheters (1.0 mm ID and 2.0 mm OD; Portex, Hythe, Kent, UK) were placed in the fetal carotid artery and jugular veins to withdraw arterial blood samples to measure pH and blood gas tensions and to infuse fluids and antibiotics, respectively. To apply CPAP to the lung, fetal trachea was cannulated with a Y-shaped endotracheal tube (4.8 mm ID and 7.9 mm OD; Tygon, Norton Plastics, Akron, OH). The endotracheal tube was advanced for 5-6 cm and transfixed (0 silk) to prevent its dislodgement from the trachea. In addition, two sutures (4-silk) were placed around the trachea to prevent air leaks during the lung distension experiments. The proximal free ends of the Y-endotracheal tubes were attached to two polypropylene tubes (4.8 mm ID, 7.8 mm OD; Tygon) that served as inspiratory and expiratory tubes. Eight fetuses underwent bilateral vagotomy while the remaining eight were sham operated. The vagus nerves were carefully separated from the carotid artery in the cervical region by blunt dissection, and 2.5- to 3.0-cmlong pieces were removed from both vagi. Because our preliminary results from vagotomized fetuses showed attenuation of FBM and it was not clear whether the attenuation of FBM was due to denervation of aortic chemoreceptors or other vagal afferents, the sham surgery included identification but not section of vagal as well as carotid sinus nerves. According to a modified technique originally described by Itskovitz and Rudolph (31), through a midline incision the superior laryngeal artery and nerves were identified and left intact. Through further blunt dissection, superior cervical ganglion and glossopharyngeal nerves were identified. The carotid sinus nerve arises from the glossopharyngeal nerve and courses from the medial aspect of the hyoid bone to the carotid body and sinus area, which are situated at the bifurcation of carotid and occipital arteries. Similar procedure was repeated on the contralateral side and the incisions were sewn in layers. This procedure added 15 min extra on each side to surgery time. Once the surgical procedures and instrumentation were complete, two amniotic catheters were sutured to the back of the fetal head, and the fetus was returned to the uterus and all maternal incisions were sewn in layers. One of the amniotic catheters (polyvinyl, 2.0 mm ID and 3.0 mm OD; Portex) was used as a reference zero value for fetal arterial blood pressure and also to monitor the amniotic pressure. The second amniotic catheter (polypropylene, 4.8 mm ID, 7.9 mm OD; Tygon) was connected to one of the two extensions of the Y-endotracheal tube to facilitate tracheoamniotic fluid flow, whereas the second extension of the endotracheal tube remained plugged when no experiments were being done.
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1403
A polyvinyl catheter (2.0 mm ID, 3.0 mm OD; Portex) was placed in the jugular vein of the ewe to administer fluids and antibiotics. The fetal electrode wires, vascular catheters, and endotracheal tube extensions were exteriorized through the left maternal flank and stored in a cloth pouch sewn adjacent to the exteriorized site. Postoperatively, the ewes received 1 g of cephalothin (Keflin, Eli Lilly Canada, Toronto, Ontario) and 80 mg of gentamicin (Cidomycin, Roussel Canada, Montreal, Quebec) intravenously and 20 mg of gentamicin into the amniotic cavity, whereas the fetuses received 150 mg of cephalothin and 10 mg of gentamicin intravenously twice daily for 5 days. Also, fetal and maternal vascular catheters were flushed twice daily with heparinized isotonic saline (10 U heparin/ml). Data acquisition and analysis. The amniotic pressure and fetal arterial blood pressure were calibrated twice daily and recorded with Statham P23 ID pressure transducers (Gould Instruments Division, Cleveland, OH). Electrophysiological signals from ECoG, EOG, EMGNK, and EMGdi were appropriately amplified and filtered (NeuroLog IOOAK, l02G, and 125/6; Medical Systems, Greenvale, NY) and, along with the amniotic pressure and blood pressure, were recorded on an eightchannel chart recorder (Gould 2800s; Gould). In addition, all signals were digitized (Neurocorder DR-886; Neurodata Instruments, New York, NY) and stored on a videocassette recorder (model FVH-C4000, Fisher). Respiratory and behavioral patterns during control and experimental conditions included manual measurements of 1) percentage of breathing time during total time (the fetus was considered to be breathing when there were at least six breaths/min, and measurements of control percentage of breathing time included equal numbers of LVand HV-ECoG periods); 2) total duration of LV-ECoG and HV-ECoG as a percentage of total time; 3) percentage of time breathing in LV-ECoG (percentage of breathing time during LV-ECoG/total LV-ECoG time); 4) percentage of time breathing in HV-ECoG (percentage of time breathing during HV-ECoG/total HV-ECoG time; both percentage of time breathing in LV-ECoG and percentage of time breathing during HV-ECoG are the most precise variables indicating the relationship between breathing and electrocortical activity); 5) single longest breathing episode in minutes (single longest breathing episode was a breathing episode where breathing was not interrupted by an apneic period); and 6) time lag to the onset of continuous breathing movements if present. Continuous breathing episodes were defined as the breathing episodes that not only remained continuous during both LV- and HV-ECoG but also terminated only when the experimental condition was changed. The diaphragm EMG signal was integrated and from the integrated signal frequency (f), number of diaphragmatic contractions per minute of breathing time, amplitude of breathing (JEMGd i ) in arbitrary units from the total area of the integrated diaphragmatic EMGdi, respiratory output ( EMGdi X f), and inspiratory time were measured. Ir hese analyses were done during arousal and rapid-eye-movement (REM) sleep in both sham-operated and vagotomized fetuses. Cardiovascular responses included measurements of systolic, diastolic, and mean
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1404 ECoG
VAGOTOMY
I
I I
I
I
I I
CONTROL
ATTENUATES
AROUSAL
1
I
I ‘t Tracheal
Suction
CPAP 3’0 cm H,O plus Randomized O2 % 0% (100% Nitrogen) 21% 50% 100% FIG. 1. Schematic representation of experimental design. First, control period consisting of breathing and nonbreathing episodes was obtained. Tracheal suction to remove tracheal fluid was followed by fetal lung distension with 4 different concentrations of 0, [0 (100% Nz), 21,50, and lOO%] at continuous positive airway pressure (CPAP) of 30 cmH,O in a randomized order. All changes were made during high-voltage electrocortical (ECoG) activity.
blood pressure and heart rate. Blood pressure was recorded continuously during control and experiments. Both blood pressure and heart rate were measured every 10 min in both sham-operated and vagotomized fetuses. The frequency response of our pressure recording system was confirmed with a Millar multifunction pressure generator (waveform generator, model WGA-200; Millar Instruments, Houston, TX) and was found to be adequate. Electrographic criteria were used to define active sleep, quiet sleep, and awake states (29, 43, 51). Active sleep was defined by the simultaneous presence of LVECoG and eye movements and absence of neck muscle tone, whereas quiet sleep was defined by the simultaneous presence of HV-ECoG and neck muscle tone and absence of eye movements. Fetal wakefulness was defined by the simultaneous presence of LV-ECoG, eye movements, and neck muscle tone. Experimental protocol. No experiments were done for at least 4 days after surgery and only after fetal physiological variables were within normal range. The normal ranges for physiological variables were the pH >7.30, arterial PCO, (PacoJ 18 Torr. The breathing time was considered normal when it was >25% of the total time. We did not record any control periods or perform any experiments during labor. The experimental design is shown in Fig. 1. The physiological variables, amniotic pressure, blood pressure, ECoG, EOG, EMGNK, and EMGdi were displayed on the chart recorder at a speed of 0.05 mm/s overnight and at variable speeds (0.05-10 mm/s) during the experiments. In addition, all control and experiments were recorded on a videocassette for future analyses of all recorded variables. Experiments were not started until 1000 h to avoid the morning trough period of FBM. Once the control period of at least 3 h was obtained, fetal trachea was suctioned through one of the endotracheal loops, the patency of both loops was checked, and fetal lungs were distended at a CPAP of 30 cmH,O, relative to atmospheric pressure, with four different concentrations of 0, [0 (100% N2), 21, 50, and lOO%] in a randomized order (Baby Bird Ventilator; Bird, Palm Springs, CA). Randomization was achieved by drawing without replacement one of the four concealed 3-in. X 3-in. pieces of papers bearing the gas concentration. This procedure was performed before each experiment. N, was added to
AND
FETAL
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MOVEMENTS
the protocol as a control for gaseous distension. The variables obtained during experiments were not compared with the overnight controls but with the controls obtained between 1000 and 1300 h on a given day. All gas mixtures were humidified and warmed to 39.5”C before administration (model 328; Fisher Paykel, Allied Products Medical Division, 25 Panmure, Auckland, New Zealand). 0, concentration was adjusted by the Baby Bird 0, blender as well as by an 0, analyzer (model 5584EC; Hudson-Ventronics Division, Temecula, CA). Each 0, concentration was maintained for -2 h, and the change in gas mixture was done only during HV-ECoG to avoid the confounding effects of fetal breathing that occur during LV-ECoG. If the fetuses continued to breathe during HV-ECoG, we waited for 1 h. However, if the fetus continued to breathe beyond that period, the change in gas concentration was made during HV-ECoG according to the randomized sequence. This step might have underestimated the duration of the single longest breathing episodes, especially among the sham-operated fetuses, but was taken to maintain consistency during the experiments. Arterial pH and blood gas tensions were analyzed during control period and during lung distension with various gas mixtures. These analyses were done during both LV- and HV-ECoG. The blood samples were kept on ice until the time of analysis, and the temperature of the blood gas analyzer was modified to 39.5OC (ILl301; Instrumentation Laboratory System, Lexington, MA). Statistical analysis. All recorded physiological variables in sham-operated and vagotomized fetuses were compared within each group as well as between the two groups. The effects of 0 (100% NJ, 21, 50, and 100% 0, on various variables were compared with the control values within each group, vagotomized and sham-operated, using analysis of variance for repeated measures. The significance of differences was assessed by Dunnett’s test. Further analysis of variance was done to investigate whether there were any differences between the two groups and whether those differences were dependent on the 0, concentration. Nonpaired t test was then done to detail these interactions between the vagotomized and sham-operated groups. Inspiratory times were compared by two-way analysis of variance. Characteristics of the fetuses (Table 1), with the exception of gestational age, were compared using Fisher’s exact test, whereas non1. Characteristics of sham-operated and vagotomized fetuses TABLE
Sham
Total no. Gestational age, days Number of oxygenated fetuses Wakefulness observed Breathing during LVand HV-ECoG
Operated
8 139+3
Vagotomized
P
8 138+2
0.652*
5
6
1.00-f
5
6
1.00-f.
5
0
0.025-f Q
Gestational age values are means + SD. LV-ECoG, low-voltage electrocortical activity; HV-ECoG, high-voltage ECoG. * Nonpaired t test; t Fisher’s exact test; $ significant difference between awake fetuses and fetuses that breathed during both LV- and HV-ECoG.
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VAGOTOMY
ATTENUATES
AROUSAL
100 s w I F g
I 2 Is!
50
m
0 6 .-
E
80
B
O-
o SHAM-OPERATED
a-
. VAGOTOMIZED
*t
$3 i2 a 40 IL z 0 0
z(r 3n
0 C
N2
21
50
100
2. Breathing time (A) and duration of arousal (B) in shamoperated and vagotomized fetuses under control conditions and during lung distension with 100% N, and 21,50, and 100% 0, at CPAP of 30 cmH,O. Breathing time decreased during lung distension with NS, whereas both breathing time and duration of arousal significantly increased during lung distension with 100% 0, in sham-operated group only. Changes in breathing time and duration of arousal were also significantly different from those observed in vagotomized fetuses. *P < 0.05 from control values within group; tP < 0.05 between sham-operated and vagotomized fetuses. FIG.
AND
FETAL
BREATHING
mized fetuses (Fig. 6). Similarly, plasma bicarbonate was 23.8 t 1.3 and 24.1 t 0.8 mM in sham-operated and vagotomized groups, whereas the base deficit was -0.9 t 1.2 and -0.7 t 1.4 mM, respectively. Although lung distension with 50% 0, in sham-operated group resulted in a small increase in Pa,, (35.0 t 16.4 Torr), it did not reach statistical significance. However, in response to 100% O,, pH decreased (P < 0.0001 and 0.036) and Paoz increased (P < 0.0001 and lOO% in blood pressure and heart rate variability among the denervated fetuses. Koos and Sameshima (35) have also reported an increased although markedly less pronounced variability in arterial blood pressure in sinoaortic-denervated fetuses. Besides the possible effects of vagotomy on pulmonary afferents as discussed above, vagal nerves also carry reflexes from chemo- and/or baroreceptors, abdominal expiratory muscles, the cardiovascular system, and abdominal viscera. Interruption of chemo- and/or baroreceptor activity can result in decreased fetal breathing movements and can also lead to respiratory failure in the newborn (11, 19, 35, 40). Therefore, this possibility needs to be investigated in our model. Bishop (6) and Bishop and Bachofen (7) showed that positive airway pressure could lead to stimulation of abdominal muscles not only during expiration but also to some extent during inspiration. Similarly, Gilmartin et al. (24) have recently shown the recruitment of various abdominal muscles during stepwise increments of positive end-expiratory pressures and vagotomy resulted in attenuation of such muscle activity. Therefore, augmentation of breathing in our studies could have been partly due to the recruitment of abdominal expiratory muscles during application of distending pressure, and such augmentation was abolished by bilateral vagotomy. In summary, we have shown that arousal and stimulation of breathing in response to lung distension and oxygenation in near-term fetal sheep can be attenuated by bilateral cervical vagotomy. Further studies involving the blood flow measurements, expiratory muscle activity, differential vagal blockade, and single-fiber recordings are warranted to dissect the mechanism(s) underlying these responses. The authors are grateful to Michelle Cavanaugh for secretarial assistance, Drs. Tak Fung and Gordon Fick for statistical analysis, and Dr. John Remmers for constructive criticism. We also acknowledge the generous financial support of the Alberta Children’s Hospital Foundation and the Alberta Lung Association. This work was presented in part at the Federation of American Societies for Experimental Biology Meeting in Washington, DC in April 1990. Address for reprint requests: S. U. Hasan, Dept. of Paediatrics, The University of Calgary, Faculty of Medicine, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Received
28 August
1991; accepted
in final
form
13 May
1992.
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AROUSAL
monary-CO, ventilation reflex in dogs: effective range of CO, and results of vagal cooling. Respir. Physiol. 34: 121-134, 1978. BARTOLI, A., B. A. CROSS, A. Guz, A. HUSZCZUK, AND R. JEFFERIES. The effect of varying tidal volume on the associated phrenic motoneurone output: studies of vagal and chemical feedback. Respir. Physiol. 25: 135-155, 1975. BISHOP, B. Abdominal muscle and diaphragm activities and cavity pressures in pressure breathing. J. Appl. Physiol. 18: 37-42, 1963. BISHOP, B., AND J. BACHOFEN. Vagal control of ventilation and respiratory muscles during elevated pressures in the cat. J. Appl. Physiol. 32: 103-112, 1972. BLANCO, C. E., C. B. MARTIN, J. RANKIN, M. LANDAUER, AND T. PHERNETTON. Changes in fetal organ flow during intrauterine mechanical ventilation with or without oxygen. J. Deu. Physiol. Oxj. 10: 53-62,1988. BLATTEIS, C. M. Hypoxia and the metabolic response to cold in new-born rabbits. J. Physiol. Lond. 172: 358-368, 1964. BODDY, K., G. S. DAWES, R. FISHER, S. PINTER, AND J. S. ROBINSON. Foetal respiratory movements, electrocortical and cardiovascular response to hypoxemia and hypercapnia in sheep. J. Physiol. Lond. 243: 599-618,1974. BOWES, G., E. R. TOWNSEND, L. F. KOZAR, S. M. BROMLEY, AND E. A. PHILLIPSON. Effect of carotid body denervation on arousal response to hypoxia in sleeping dogs. J. Appl. Physiol. 51: 40-45, 1981. BRADLEY, G. W., M. I. M. NOBLE, AND D. TRENCHARD. The direct effect of pulmonary stretch receptor discharge produced by changing lung carbon dioxide concentration in dogs on cardiopulmonary bypass and its action on breathing. J. Physiol. Lond. 261: 359-373, 1976. BUREAU, M. A., J. LAMARCHE, P. FOULON, AND D. DALLE. Postnatal maturation of respiration in intact and carotid body-chemodenervated lambs. J. Appl. Physiol. 59: 869-874, 1985. COLERIDGE, H. M., AND J. C. G. COLERIDGE. Reflexes evoked from tracheobronchial tree and lungs. In: Handbook of Physiology. The Respiratory System. Control ofBreathing. Bethesda, MD: Am. Physiol. Sot., 1986, sect. 3, vol. II, pt. 1, chapt. 12, p. 395-430. COOMBS, H. C., AND F. H. PIKE. The nervous control of respiration in kittens. Am. J. Physiol. 95: 681-693, 1930. CROSS, B. A., P. W. JONES, AND A. Guz. The role of vagal afferent information during inspiration in determining phrenic motoneurone output. Respir. Physiol. 39: 149-167, 1980. DAWES, G. S., H. E. Fox, B. M. LEDUC, G. C. LIGGINS, AND R. T. RICHARDS. Respiratory movements and rapid eye movement sleep in the fetal lamb. J. Physiol. Lond. 220: 119-143, 1972. DIMARCO, A. F., C. VON EULER, J. R. ROMANIUK, AND Y. YAMAMOTO. Positive feedback facilitation of external intercostal and phrenic inspiratory activity by pulmonary stretch receptors. Acta Physiol. Stand. 113: 375-386, 1981. DONNELLY, D. F., AND G. G. HADDAD. Prolonged apnea and impaired survival in piglets after sinus and aortic nerve section. J. Appl. Physiol. 68: 1048-1052, 1990. FEDORKO, L., E. N. KELLY, AND S. J. ENGLAND. Importance of vagal afferents in determining ventilation in newborn rats. J. Appl. Physiol. 65: 1033-1039, 1988. FINER, N. N., I. F. ABROMS, AND H. W. TAEUSCH. Ventilation and sleep states in newborn infants. J. Pediatr. 89: 100-108, 1976. FINK, B. R. Influence of cerebral activity in wakefulness on regulation of breathing. J. Appl. Physiol. 16: 15-20, 1961. FOUTZ, A. S., A. NETICK, AND W. C. DEMENT. Sleep state effects on breathing after spinal cord transection and vagotomy in the cat. Respir. Physiol. 37: 89-100, 1979. GILMARTIN, J. J., V. NINANE, AND A. DE TROYER. Abdominal muscle use during breathing in the anesthetized dog. Respir. Physiol. 70: 159-171, 1987. HADDAD, G. G., AND R. B. MELLINS. The role of airway receptors in the control of respiration in infants: a review. J. Pediatr. 91: 281286, 1977. HARDING, R., D. J. HENDERSON-SMART, P. JOHNSON, AND M. E. MCCLELLAND. Posturally related tonic activity recorded from the peripheral diaphragm in awake and sleeping lambs (Abstract). J. Physiol. Lond. 292: 57, 1979. HASAN, S. U., AND A. RIGAUX. The effects of lung distension, oxygenation, and gestational age on fetal behavior and breathing movements in sheen. Pediatr. Res. 30: 193-201. 1991.
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