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Respiratory neural activity responses to chemical stimuli in newborn rats: reversible transition from normal to ‘secondary’ rhythm during asphyxia and its implication for ‘respiratory like’ activity of isolated medullary preparation
Neuroscience Research 38 (2000) 407 – 417 www.elsevier.com/locate/neures
Respiratory neural activity responses to chemical stimuli in newborn rats: reversible transition from normal to ‘secondary’ rhythm during asphyxia and its implication for ‘respiratory like’ activity of isolated medullary preparation Yasuichiro Fukuda * Department of Physiology II, School of Medicine, Chiba Uni6ersity, 1 -8 -1 Inohana, Chuo-ku, Chiba City 260 -8670, Japan Received 26 June 2000; accepted 22 August 2000
Abstract To clarify a possible origin of ‘respiratory like’ rhythmic activities observed in in vitro brainstem preparation, the phrenic (Phr) and cranial nerve (XII or IX) inspiratory activities were analyzed in halothane-anesthetized, vagotomized and artificially ventilated newborn (2–6 days after birth) and young adult rats (30 – 50 days) during altered chemical stimuli and prolonged asphyxia at 25°C. The newborn rat showed regular rhythmic inspiratory discharges of short duration, and their responses to CO2 and hypoxia did not differ from those seen in adult rats. In the newborn rat the Phr and cranial nerve inspiratory discharges increased first, then respiratory frequency decreased and finally ceased completely for 1 – 2 min during asphyxia. Thereafter, ‘secondary’ rhythmic inspiratory activity emerged at a slower rate with decremental inspiratory discharge profile, which persisted for a period more than 40 min of asphyxia. A normal respiratory activity recovered after resumption of artificial ventilation. Though young adult rats exhibited similar sequential changes in respiratory activity during asphyxia, the ‘secondary’ rhythmic activity persisted for a period of several min only. The pattern of ‘secondary’ respiratory activity corresponded well with that of rhythmic activities seen in the isolated medullary block preparation of newborn rat. ‘Respiratory like’ activity found in isolated medullary preparations of newborn animals may arise from a mechanism that generates ‘secondary’ (or so called ‘gasping’ type) rhythmic inspiratory activity during prolonged asphyxia in in vivo preparations. © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Newborn rat; Phrenic and cranial nerve inspiratory activity; Normal respiratory rhythm; ‘Secondary’ respiratory rhythm; Asphyxia
1. Introduction Recent observations on the isolated brainstem (spinal cord) or medullary block/slice preparations obtained from newborn animals have provided essential evidence for a possible site(s) and cellular mechanisms underlying respiratory rhythm generation (Suzue, 1984; Smith et al., 1995; Onimaru et al., 1997). Onimaru and his group (Onimaru et al., 1987, 1988) found that the propriobulbar neurons showing pre-inspiratory dis-
* Tel.: +81-43-2262030; fax: + 81-43-2262034. E-mail address: [email protected] (Y. Fukuda).
charge pattern in the rostro-ventrolateral medulla involve primary rhythm generating mechanism. Smith et al. (1995) have hypothesized that the pre-Bo¨tzinger complex, a site within the ventrolateral medulla, contains the neuron population initiating a primary rhythm of respiration. These medullary neurons and their ‘respiratory like’ rhythmic activity found in in vitro preparations may also have functional role in the generation of normal respiratory rhythm in in vivo animals. Smith et al. (1990) identified rhythmic neural discharges of isolated brainstem spinal cord preparation with inspiratory discharges in vivo animals at a comparable experimental condition in the newborn rat. However, critical arguments have confronted such favorable interpretation. These include that output mo-
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toneurons of in vitro preparation display always ‘gasping’ type discharge and no one has reported normal respiratory activity pattern. There is a general agreement that substantial analog of in vitro ‘respiratory like’ activity should be found in in vivo preparation during altered chemical stimuli particularly during anoxia or asphyxia. Wang et al. (1996) comparing inspiratory discharge patterns between in vivo newborn rats during anoxia and in vitro preparations, stated that mechanism and site of generation of in vitro ‘gasping’ type activity differ from that of normal respiratory pattern. If this were the case (St John, 1996), two rhythm generators of different origins (‘gasping’ and normal types) would drive common outputs such as phrenic and cranial nerve motoneurons. However, validity of such two rhythm generator concept still requires further analysis of not only individual inspiratory burst profile but also changes in rhythmicity, temporal aspect of transition from normal to ‘gasping’ rhythms during anoxia and their reversibility. Concerning the respiratory rhythmicity during anoxia or asphyxia, ventilatory measurements in newborn animals by previous many studies (see review, Thach et al., 1991) have already established a sequence of changes in spontaneous ventilatory or air flow pattern in response to asphyxia (or anoxia): initial hyperpnea, primary apnea, hypoxic (or asphyxic) ‘gasping’ and secondary apnea. Asphyxic ‘gasping’ functions as an important autoresuscitation process for survival in newborn animals by which these animals show particular tolerance to anoxia and asphyxia (Thach et al., 1991). However, correlative neurophysiological analysis is required to confirm these findings. Perhaps asphyxic ‘gasping’ observed in in vivo neonates corresponds to respiratory like rhythmic activity found in in vitro preparation. These conflicting interpretations of the results depend presumably upon that respiratory neural activities of newborn rat have not been analyzed systematically and quantitatively at controlled levels of chemical stimuli or during asphyxia. The present study aims to analyze the phrenic and cranial nerve (XII or IX) inspiratory discharge responses to altered CO2 and hypoxic stimuli during eupnea and to prolonged asphyxia. Experiments were designed to observe sequential changes in phrenic and cranial nerve inspiratory activities during progression of asphyxia not only in the newborn but also in the young adult rats. The respiratory neural activities during asphyxia were further compared with rhythmic activity in in vitro medullary block preparations obtained from newborn rats. The results revealed a reversible shift of normal respiratory rhythmicity to a more slower ‘secondary’ (or ‘gasping’) respiratory rhythm during prolonged asphyxia. The latter coincided well with ‘respiratory like’ rhythmic activity found in isolated medullary block preparation.
2. Material and methods
2.1. General We used newborn rats (3–6 days: 5–10 g) and young adults rats (30–50 days: 110–160 g) of either sex. Following anesthetized with halothane (induction 2.0– 2.5%, maintenance during surgery 1.5–1.2%), animals were tracheotomized and artificially ventilated by a rodent respiratory at a rate of 80–100/min with a constant stroke volume (0.8–1.5 ml for newborns, 1.5– 2.5 ml for young adults) (Shinano Seisakusho, type 170, Tokyo) under subcutaneous administration of a muscle relaxant (Mioblock® (2 mg/ml), total 0.1 ml/body in the newborn and 0.8 ml/body in young adults). We sectioned bilateral vagus nerves at the mid cervical region. In the control condition body temperature was maintained at 37°C by a heating pad, a temperature regulator and a thermistor probe (tip diameter 1 mm) placed in the rectum. Intra-tracheal gas was continuously sampled to measure fractional concentration of CO2 and O2 (FCO2, FO2) by a gas analyzer (25 ml/min, NEC-Sanei Instruments, type 1H26, Tokyo). The tracheal peak FCO2 and the inspiratory FO2 values were maintained at about 0.07–0.08 and above 0.50, respectively, in the control condition by adjusting the amount of CO2 and O2 gas flowing into the inspiratory line. The phrenic and hypoglossal (or glossopharyngeal) (XII or IX) nerves were separated from surrounding tissues and dissected for recording inspiratory activity by a bipolar Pt–Ir electrode in liquid paraffin/mineral oil mixture. Although the onset and fading of respiratory rhythmic activity are best detected by inspiratory discharge of IX nerve (Fukuda et al., 1995), recording of IX nerve activity was sometimes difficult in in vivo and in vitro newborn rats. We, therefore, analyzed inspiratory burst duration of Phr and XII nerves. Original neural activity and its leaky integrated wave were recorded on a chart recorder and were stored on magnetic tape for later analysis. We also recorded ECG as an index of cardiac function.
2.2. Reduction of body temperature To compare rhythmic activities between in vivo whole animal and in vitro preparation at the same temperature condition, the rectal temperature was decreased from 37 to 25°C by cooling the body surface as described previously (Maruyama and Fukuda, 2000). This was because in vitro brainstem (-spinal cord or medullary block) preparations have been examined at lower temperature (24–27°C) to maintain viability of isolated brain tissue. Due to significant temperature effects on anesthesia (Pavlin and Hornbein, 1986) the depth of anesthesia at 25°C was maintained at an almost similar level of ‘depth’ with that in control 37°C
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by decreasing inspiratory halothane concentration from 1.1% in normothermia to 0.4 – 0.5% during hypothermia (25°C) (Maruyama and Fukuda, 2000). This adjustment of halothane concentration was critically important for the analysis of respiratory control mechanism in response to chemical stimuli during induced hypothermia (Regan and Eager, 1966).
2.3. Effect of changing chemical stimuli and asphyxia The effect of CO2, hypoxia and asphyxia on Phr and XII (or IX) inspiratory discharges was examined at 25°C. The level of CO2 stimulus was altered by changing the amount of CO2 injected into the inspiratory line with monitoring peak tracheal FO2 value. We also tested effects of step wise reduction in inspiratory FO2 (FIO2) from above 0.50 down to 0.05. Effects of asphyxia were examined as follows. After recording respiratory nerve activity in the control hyperoxic (FIO2 \0.50) and slightly hypercapnic condition (peak FCO2 =0.07 – 0.08%) at 25°C, artificial ventilation was ceased for more than 50 min in the newborn and about 10 min in the young adult rats. Changes in Phr and XII (or IX) inspiratory activities were continuously recorded during cessation of artificial ventilation and during recovery after resumption of artificial ventilation.
2.4. Isolated medullary block preparation of newborn rat Seven newborn rats (2 – 6 days) of either sex were deeply anesthetized with halothane. The lower brainstem was quickly isolated and placed immediately in a cold mock CSF (4°C). A transverse section of medullary region from a level of obex and the lower edge of trapezoid body (thickness 1.5 mm) was made manually with using a steel razor blade in cold mock CSF (4°C), and the tissue block was placed in a continuously flowing mock CSF in an incubation chamber at 25°C. The mock CSF had following composition in mM: NaCl 135, KCl 4.0, CaCl2 1.3, MgSO4 0.6, NaH2PO4 0.6, NaHCO3 24 and glucose 20, and the solution had pH of 7.40 when equilibrated with 95%O2 – 5%CO2 gas mixture. The ‘respiratory like’ activity was monitored from the XII rootlet with a suction electrode (tip internal diameter 0.05 mm), and a unit activity was simultaneously recorded from the ventral medullary region by a glass capillary microelectrode (tip diameter 2 mm). We compared the rate of rhythmic activity and individual (inspiratory) burst pattern between in vivo preparation during asphyxia and in vitro preparations.
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2.5. Data analysis Normal respiratory frequency, Phr and XII inspiratory duration was compared between young adult and newborn rats during hyperoxic breathing at 37 and 25°C. The rate of ‘secondary’ rhythmic activity and inspiratory bursts duration during asphyxia were compared with that obtained in hyperoxia at 25°C. Furthermore, the rate of rhythmic activity was compared quantitatively between in vivo whole animal preparations during prolonged asphyxia and in vitro medullary block preparations. We considered statistically significant difference (PB 0.05) of compared data by ANOVA and Bonferroni correction of t-test.
3. Results
3.1. Comparison of phrenic and cranial ner6e inspiratory acti6ities between newborn and young adult rats Fig. 1 compares inspiratory activities of the Phr and cranial nerves (IX or XII) between newborn and young adult rats at control condition (hyperoxia, peak tracheal FCO2 0.07–0.08, 37°C). Although the rate of rhythm (respiratory frequency) did not differ between newborn and young adult rats, newborn rats showed short duration of Phr and XII burst (Table 1, Fig. 1). With reduction in body temperature from 37 to 25°C, respiratory frequency decreased and the duration of the Phr and XII nerve inspiratory bursts prolonged significantly in both young adult and newborn rats (Table 1, Fig. 2). In the newborn rat, each Phr or cranial nerve inspiratory burst often consisted of a train of several short bursts. The newborn rat showed significantly slower respiratory frequency and shorter duration of Phr and XII inspiratory bursts than the young adult rat at 25°C in hyperoxia (Table 1).
3.2. Inspiratory acti6ity response to chemical stimuli in the newborn rat The newborn rats showed almost similar responses of Phr and XII inspiratory discharge to changing the level of chemical stimuli as usually seen in normothermic adult rats. Respiatory frequency and inspiratory discharges of newborn rats increased with elevating tracheal FCO2, and respiratory rhythmicity ceased during hypocapnia (hypocapnic apnea) (Fig. 3). At a higher peak FCO2 (above 0.08), however, respiratory frequency rather decreased (Fig. 3D). Effects of decreasing FIO2 are shown in Fig. 4. Respiratory frequency and inspiratory discharges increased as decreasing FIO2 from 0.40 to 0.08 accompanying with rapid onset of Phr activity. Furthermore, the onset of cranial nerve activity became
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Fig. 1. Respiratory neural activity of halothane anesthetized, vagotomized and artificially ventilated young adult (A) and newborn rats (B,C) at 37°C in hyperoxia. (A) Young adult rat (30 days); (B) newborn rat (3 days); (C) newborn rat (2 days). Paw, airway pressure; FCO2, fractional concentration of CO2 in tracheal gas; Phr, phrenic nerve activity; Phr, integrated phrenic nerve activity; IX, glossopharyngeal nerve activity; integrated glossopharyngeal nerve activity; XII, hypoglossal nerve activity; XII, integrated hypoglossal nerve activity. These abbreviations are the same in the following figures.
Table 1 Respiratory neural activity of newborn and young adult rat at 37°C (hyperoxia, normal rhythm), 25°C (hyperoxia, normal rhythm), and during asphyxia (‘secondary’ rhythm) in in vivo whole animal preparations and in isolated medullary block preparations (newborn rat only) Condition
n
f (min−1)
Newborn rats (in 6i6o) 37°C, hyperoxia (normal rhythm) 25°C, hyperoxia (normal rhythm) 25°C, asphyxia (‘secondary’ rhythm)
(8) (8) (8)
31.8 95.8 16.3 93.1* 5.6 91.0 c
Newborn rats (isolated medullary preparation)
(7)
6.5 90.8
Young adult rats (in 6i6o) 37°C, hyperoxia (normal rhythm) 25°C, hyperoxia (normal rhythm) 25°C, asphyxia (‘secondary’ rhythm)
(7) (7) (7)
31.3 95.0 26.0 93.7* 18.3 93.0 c ,++
Phr duration (ms)
188 9 64 526 9189* 320 9 122 c
XII duration (ms)
394 9 138 822 9312* 285 987 c 464 956+
669 9141 1092 9 137* 407 9152 c
910 9 95 1448 9225* 289 9 84 c
* Statistically significant difference (PB0.05) between 37 and 25°C in hyperoxic conditions. c Statistically significant difference (PB0.05) between hyperoxic and asphyxic conditions at 25°C. + Statistically significant difference (PB0.05) between in vivo asphyxic and in vitro isolated preparations at 25°C (newborn rat only). ++ Statistically significant difference (PB0.05) between newborn and young adult rats at 25°C during asphyxia.
Fig. 2. Effect of decreasing body temperature from 37 (A) to 25°C (B) on respiratory nerve activity in newborn rat (5 days).
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Fig. 3. Effect of changing CO2 stimulus on respiratory nerve activity in newborn rat (4 days) during hyperoxia (FIO2 \0.50) at 25°C.
Fig. 4. Effect of changing FIO2 on respiratory nerve activities in newborn rat (2 days) during isocapnia (peak FCO2, 0.06) at 25°C.
Fig. 5. Antagonistic effect of hypocapnic (peak FCO2 0.04) inhibition and hypoxic stimulation (FIO2 0.08) on respiratory nerve activities in newborn rat (4 days) at 25°C.
earlier and the duration of inspiratory bursts prolonged greatly. However, respiratory frequency determined by rhythmic Phr activity decreased with further reduction in FIO2 to 0.05 (Fig. 4). Some cranial nerve bursts did not accompany with synchronized Phr burst. As shown in Fig. 5, when respiratory rhythmicity ceased during
hypocapnia (peak FCO2 0.03–0.04), the reduction in FIO2 (0.08) induced regeneration of rhythmicity and augmented the inspiratory discharges. Such counteracting effect of hypoxia and hypocapnia was completely similar to those usually seen in adult rats at normothermia (figure not shown).
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Fig. 6. Effect of prolonged asphyxia on respiratory nerve activities in newborn rat (3 days) at 25°C. (A)–(D) were obtained from the same rat before and during asphyxia. Asphyxia was produced by cessation of artificial ventilation. (B) was obtained at 1 min after (A), (C) was 10 s after (B), and (D) was 30 min after (C).
3.3. Inspiratory acti6ity during asphyxia and reco6ery 3.3.1. Newborn rats Following cessation of artificial ventilation, respiratory frequency and the magnitude of integrated Phr and cranial nerve activity increased gradually, then fre-
quency decreased with prolongation of expiratory time, and finally respiratory rhythm expired completely for 1–2 min (primary apnea, Fig. 6). Individual inspiratory burst pattern of Phr and XII nerves changed from normal augmenting to abrupt onset with long lasting decremental pattern just before cessation of normal rhythm (Fig. 7). The onset of cranial nerve inspiratory activity became much earlier initially and then became almost simultaneous before cessation of rhythm. Normally within several minutes of apnea, a new rhythmic activity appeared first in the cranial nerves which was later followed by the appearance of Phr inspiratory activity. We defined such new rhythm as ‘secondary’ respiratory rhythm. At this initial stage, the onset of cranial nerve inspiratory activity preceded the start of Phr discharge as seen before asphyxiation. The Phr inspiratory activity showed an abrupt onset and decremental decay, which was indistinguishable from those seen before cessation of normal rhythm (compare ((2), (3)) and ((5), (6)) in Fig. 7). During a later steady state condition, the onset of inspiratory activity became almost simultaneous in both the Phr and cranial nerves (Fig. 7), and each inspiratory activity showed abrupt onset with decremental firing pattern. These synchronized rhythmic Phr and cranial nerve inspiratory activities persisted for a period more than 40 min of asphyxia. At that time there was neither QRS wave in the ECG tracing (figure not shown) nor visible pulsation in the carotid artery suggesting almost total arrest of arterial circulation. As shown in Table 1, the rate of ‘secondary’ rhythmic activity at steady state condition (20–40 min asphyxia) was significantly slower than that of normal rhythm observed during hyperoxic control condition. There was no significant difference in the duration of Phr and XII inspiratory burst duration between normal and ‘secondary’ rhythmic activities during asphyxia. The rate of secondary rhythm and amplitude of integrated Phr or XII inspiratory discharges decreased gradually and finally be came undetectable (final apnea) at 60–80 min asphyxia.
Fig. 7. Shape of individual inspiratory discharge profile at various time before and during asphyxia indicated by numbers at the top of each trace in Fig. 6.
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Fig. 8. Recovery of respiratory activity after prolonged asphyxia in newborn rat (3 days) at 25°C. (A) – (C) were obtained from the same animal. (A) Before asphyxia; (B) during asphyxia and recovery; (C) 5 min after (B). Individual inspiratory burst profile at various time indicated by number and allow is shown in higher chart speed recording in lower trace.
Following resumption of artificial ventilation at 40– 50 min of asphyxia, ECG activity (QRS wave) and carotid pulsation recovered first (figure not shown). The ‘secondary’ rhythmic activity ceased for a while (1–3 min) (Fig. 8B), thereafter respiratory rhythm recovered initially at a slow rate and later became faster and finally returned to pre-asphyxic level (Fig. 8C). Shape of individual inspiratory bursts recovered also. When asphyxia test was repeated, normal rhythm shifted smoothly to secondary rhythm without interruption by clear primary apnea period. Normal or ‘secondary’ respiratory rhythmic activities did not recover with resumption of artificial ventilation when duration of asphyxia exceeded 90 min.
3.3.2. Young adult rats Typical example of changes in respiratory activity during asphyxia in the young adult rat is presented in Fig. 9. After cessation of artificial ventilation, inspiratory discharges of Phr and cranial nerves increased initially, then became tonic and finally ceased completely for 2–3 min. ‘Secondary’ rhythmic activity appeared first in the Phr nerve which was later followed by small synchronized cranial nerve inspiratory discharges. The pattern of’secondary’ rhythmic activity during asphyxia differed from that in hyperoxic pre-asphyxic condition (Fig. 9 and Table 1). The respiratory frequency of asphyxic ‘secondary’ rhythm was significantly lower than that during hyperoxia. The Phr inspiratory activity during ‘secondary’ rhythm was gradual in onset reaching peak with gradual decline. These ‘secondary’ respiratory activities became slower and smaller and terminated within several min (Fig. 9), which was associated with circulatory arrest. Neither
respiratory rhythmic activities nor ECG activities recovered even with resumption of artificial ventilation at 5–6 min asphyxia (Fig. 10).
3.4. Pattern of rhythmic acti6ity in isolated medullary block preparation of newborn rat Rhythmic mass discharge was consistently recorded from the XII nerve root in the medullary block preparation of newborn rat. The rhythmic XII activity fired synchronously with burst of rhythmic unit activity recorded from rostro-ventral medullary region and its vicinity (Fig. 11). There was no significant difference between the rate of rhythmic activity in isolated medullary preparations and that of ‘secondary’ respiratory activity in in vivo preparation during asphyxia (Table 1). Furthermore, , decremental discharge pattern of the XII nerve activity in isolated preparation was almost analogous to that of the XII inspiratory discharge of in vivo new born rat during steady state asphyxic condition (Fig. 11).
4. Discussion We have recorded successively respiratory nerve discharges and their responses to chemical stimuli in the newborn rat. A high respiratory frequency and short duration of Phr and XII (or IX) inspiratory bursts characterized a feature of respiratory neural pattern of newborn rat during hyperoxic control condition. The newborn rats showed almost similar respiratory nerve activity responses to CO2 and hypoxia as seen in normal adult rat. Therefore chemical control mechanisms
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Fig. 11. Comparison of asphyxic ‘secondary’ respiratory activity of in vivo rat (4 days) (A) and rhythmic activity observed in in vitro medullary block preparation (rostro-ventral medullary region) of newborn rat (3 days) (B) at 25°C. (A) and (B) were obtained from different animals.
of respiratory system have, at least qualitatively, well developed at 2–6 days after birth. The present experiments demonstrated following three results. First, pattern of respiratory motoneuron discharges shifted from normal to a slower ‘secondary’ rhythmic type during prolonged asphyxia, and normal respiratory pattern recovered after resumption of artificial ventilation in newborn rats. Second, young adult rat exhibited also a similar ‘secondary’ respiratory activity during asphyxia but for a short period only. Third, pattern of’secondary’ respiratory activity was indistinguishable from rhythmic activity found in in vitro medullary block preparation of newborn rat. Fig. 9. Effect of prolonged asphyxia on respiratory nerve activities in young adult rat (35 days) at 25°C. (A)–(D) were obtained from the same animal. (A) and (B) were continuous recordings, (C) was obtained 6 min after (B). Shape of individual inspiratory burst profile at time indicated by number is shown in (D) with higher chart speed recording.
4.1. Respiratory pattern change during asphyxia Prolonged asphyxia in the newborn rat initiated a sequence of change in respiratory activity, i.e., augmentation of Phr and XII (or IX) inspiratory activities, cessation of rhythmicity (primary apnea), generation of
Fig. 10. Effect of asphyxia on respiratory nerve activities in young adult rat (30 days) at 25°C. (A) and (B) were obtained from the same animal. (B) was 1 min after (A). Note that secondary rhythmic activity disappeared and normal rhythmic activity did not recover even after resumption of artificial ventilation.
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‘secondary’ rhythm and final apnea. These systematic alterations correlated well with previous observations on ventilatory pattern change during asphyxia (hyperpnea, primary apnea, hypoxic ‘gasping’ and secondary apnea) (Thach et al., 1991). Although the term ‘gasp’ has been used in reference to the last respiratory pattern appearing prior to death in the course of asphyxiation, ‘gasp’ has now been viewed as coordinated and functional respiratory activity (Thach et al., 1991). We, therefore, used ‘secondary’ respiratory activity instead of ‘gasp’. In neurophysiological studies ‘gasp’ type activity during hypoxia is characterized by strong inspiratory activity with abrupt onset and gradual decay (decremental pattern) (St John, 1996). However, as seen in Fig. 4, Phr inspiratory discharge profile became abrupt in onset and with gradual decay during graded reduction in FIO2 (Fig. 4, FIO2 0.05) and also during an early phase of asphyxia even during continuation of normal respiratory rhythm (Fig. 7). Discharge profile of individual inspiratory bursts alone, i.e. decremental discharge pattern, thus, represents neither alterations of rhythm generation nor specific feature of ‘gasping’ type respiratory rhythm, but shows the presence of strong hypoxic (and hypercapnic) drive to inspiratory output motoneurons. Normal and ‘secondary’ respiratory rhythm differed significantly in their rate of rhythmicity (Table 1), which suggested a possibility that two rhythm generators of different origin with different inherent rhythmicity could drive common Phr and XII (or IX) motoneurons. Because normal respiratory activity recovered after resumption of artificial ventilation, the mechanism generating normal respiratory rhythm had not been deteriorated but rather suppressed functionally during prolonged asphyxia. A reversible hypoxic inhibition of respiratory activity has been well known and seen particularly in newborn or young animals (Haddad et al., 1982; Mortola, 1993; Fukuda, 1992). Accumulation of inhibitory substances such as adenosine, opioids, GABA accounts for occurrence of hypoxic respiratory inhibition (Neubauer et al., 1990), particularly of respiratory frequency or rhythm generation (Fukuda, 1991), which leads to the cessation of normal rhythm (primary apnea). A simple explanation is that following hypoxic inhibition of normal rhythm generation a separate mechanism of initiating ‘secondary’ rhythm becomes active to drive common inspiratory outputs, i.e. Phr or XII motoneurons. St John and Knuth (1981), St John (1996) have hypothesized separate sites of ‘gasp’ and normal (eupneic) respiratory medullary centers. This hypothesis necessarily requires an assumption that mechanism of ‘secondary’ (or ‘gasping’) rhythm is inactive or inhibited in hyperoxic condition. Another possibility may include that the rate of rhythmicity of normal rhythm generator in the medulla is changed by deprivation of inputs from other neural structures during prolonged asphyxia (see review,
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Thach et al., 1991). This case does not require a separate ‘gasp’ center in the medulla oblongata. Further analyses are necessary on transition mechanism(s) from normal to ‘secondary’ rhythms and it neuroanatomical correlates. In the present study we did not analyze correlation between circulatory and respiratory alterations during asphyxia. Thach et al. (1991) indicated possible importance of cardiovascular function for maintenance of gasping. However, the fact that ‘secondary’ respiratory activity during asphyxic condition was seen under almost total circulatory arrest suggested that generation of ‘secondary’ respiratory activity might have appeared without obvious dependency on intact medullary blood supply.
4.2. Comparison between newborn and young adult rats Young adult rats showed similar sequential change in respiratory activity during asphyxia as in newborn rats, i.e. respiratory stimulation followed by suppression leading to apnea, generation of ‘secondary’ respiratory rhythm and final apnea. The average rate of ‘secondary’ rhythm was significantly slower than that during control condition as in newborn rats. However, the duration of ‘secondary’ rhythm was much shorter than that of newborn rats (10 min vs. more than 40 min). Yuan et al. (1997) also described that 1-day-old rats gasped much longer than the 8-day-old rats. Much shorter period of displaying asphyxic ‘secondary’ respiratory rhythm and a lack of recovery of respiratory rhythm after resumption of artificial ventilation in the young adult rat may be due to irreversible damages of neural mechanism generating respiratory rhythm during prolonged asphyxia. Jacobi and Thach (1989) studied the effect of maturation on the ability of autoresuscitation by ‘gasping’ in mice and found that decrease in survival in older mice. Tolerance to hypoxia decreases gradually during early developmental phase after the birth (Duffy et al., 1974). Altered vulnerability of neural tissues to anoxia depends upon changes in metabolic states, i.e. transition from predominance of anaerobic to aerobic during development (see review, Nioka and Chance, 1991). The neuronal mechanism initiating ‘gasp’ may depend largely upon anaerobic metabolism (Tschirgi and Gerard, 1947). Yuan et al. (1997) found that hyperglycemia prolongs the duration of anoxic ‘gasping’ period in the newborn rats of 8 days age suggesting glycolytic energy supply for generation of ‘gasping’ type respiratory activity. The differences in asphyxic tolerance due to postnatal age involve various O2 and metabolic related factors such as hemoglobin’s O2 affinity, O2 consumption, O2 availability, glycogen storage and/or mitochondrial capability (Nioka and Chance, 1991). In the young adult rats phrenic inspiratory bursts during ‘secondary’ rhythm was gradual in onset reach-
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ing peak followed by gradual decay, which contrasted to abrupt onset and decremental decay in the newborn rat. This finding again suggested the pattern of individual inspiratory bursts may not characterize particular feature of asphyxic ‘secondary’ (or ‘gasping’) respiratory activity.
4.3. Comparison of rhythmic (respiratory) acti6ity between in 6i6o and in 6itro preparations Despite completely different experimental approaches, the rate and shape of rhythmic activity did not differ between in vivo preparation during asphyxia and in in vitro preparations. A plausible explanation may be that anoxia tolerant mechanism of generating ‘secondary’ (or ‘gasping’ type) respiratory rhythmicity had been still active and functioned even in in vitro medullary preparations. Suzue (1984), in his pioneer study, already suggested the rhythmic pattern of in vitro brain stem spinal cord preparation may be ‘gasping’. Absence of ‘normal’ respiratory like rhythmic activity in in vitro preparation is due either to a lack of enough tissue oxygenation to activate its generating mechanism even by superfusion with hyperoxic mock CSF or to irreversible tissue damages in isolated condition. However, in vitro rhythmic activities were recorded from medullary block preparations superfused with hyperoxic and normocapnic solution while ‘secondary’ respiratory activity was seen during as phyxia, i.e. hypoxic and hypercapnic condition. Although local PO2 and pH values in deeper medullary tissues have been shown to be lower in brain-stem spinal cord preparations (Okada et al., 1993), these were not measured in the present experiment. Tissue PCO2 and PO2 of isolated medullary block preparations should be measured in future studies. Effects of hypoxia and/or hypercapnia may also be examined in isolated medullary preparation. Recent observation on the isolated medullary slice of mice showed that normal respiratory, sighs or gasping type activities are all recorded from a single medullary area, preBo¨tzinger region under application of high KCl (8 mM) in superfusing solution and with application of synaptic inhibitors (Lieske et al., 2000). A comparison between in vivo and in vitro preparations is required in mice. Further experiments are also necessary on in vivo newborn animals in which the medulla oblongata is isolated totally from surrounding tissues to remove influences from higher centers or from peripheral afferents including vagal and carotid chemoreceptor afferents. Although St John et al. (1984) showed that gasp-like breathing in normoxic and brain stem lesioned cats is little affected by carotid chemoreceptor afferents, roles of peripheral chemoreceptor afferents in the generation of asphyxic ‘secondary’ (or ‘gasping’) rhythmic activity have not been elucidated (Thach et al., 1991).
References Duffy, T.E., Kohle, S.J., Vannucci, R.C., 1974. Carbohydrate and energy metabolism in perinatal rat brain: relation to survival in anoxia. J. Neurochem. 24, 271 – 276. Fukuda, Y., 1991. Hypoxic inhibition of respiratory neural regulation in anesthetized rats. Jpn. J. Physiol. 41, 891 – 906. Fukuda, Y., 1992. Changes in ventilatory response to hypoxia in the rat during growth and aging. Pflu¨gers Arch. 421, 200 – 203. Fukuda, Y., Tanaka, K., Chiba, T., 1995. Inspiratory activity responses to lung inflation and ventral medullary surface cooling of glossopharyngeal nerve (stylopharyngeal muscle branch) and its motoneuron distribution in the rat. Neurosci. Res. 23, 103–114. Haddad, G.G., Gandhi, M.R., Millins, R.B., 1982. Maturation of ventilatory response to hypoxia in puppies during sleep. J. Appl. Physiol. 52, 309 – 314. Jacobi, M.S., Thach, B.T., 1989. Effect of maturation on spontaneous recovery from hypoxic apnea by gasping. J. Appl. Physiol. 66, 2384 – 2390. Lieske, S.P., Thoby-Brisson, M., Telgkamp, P., Ramirez, J.M., 2000. Reconfiguration of the neural network controlling multiple breathing patterns: eupnea, sighs and gasps. Nat. Neurosc. 3, 600 – 607. Maruyama, R., Fukuda, Y., 2000. Ventilation- and carotid chemoreceptor discharge-response to hypoxia during induced hypothermia in halothane anesthetized rat. Jpn. J. Physiol. 50, 91 – 99. Mortola, J.P., 1993. Hypoxic hypometabolism in mammals. News Physiol. Sci. 8, 79 – 82. Neubauer, J.A., Mellon, J.E., Edelman, N.H., 1990. Modulation of respiration during brain hypoxia. J. Appl. Physiol. 68, 441–451. Nioka, S., Chance, B., 1991. Energy metabolism and ionic homeostasis during hypoxic stress in the developing mammalian brain. In: Haddad, G.G., Farber, J.P. (Eds.), Developmental Neurobiology of Breathing, Lung Biology in Health and Disease, vol. 53. Marcel Dekker, New York, pp. 615 – 641. Okada, Y., Mu¨ckenhoff, K., Hotermann, G., Acker, H, Scheid, P., 1993. Depth profile of pH and PO2 in the isolated brain stemspinal cord of the neonatal rat. Respir. Physiol. 93, 315–326. Onimaru, H., Arata, A., Homma, I., 1987. Localization of respiratory rhythm-generating neurons in the medulla of brainstemspinal cord preparations from newborn rats. Neurosc. Lett. 78, 151 – 155. Onimaru, H., Arata, A., Homma, I., 1988. Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat. Brain Res. 445, 314 – 324. Onimaru, H., Arata, A., Homma, I., 1997. Neuronal mechanisms of respiratory rhythm generation: an approach using in vitro preparation. Jpn. J. Physiol. 47, 385 – 403. Pavlin, E.G., Hornbein, T.F., 1986. Anesthesia and the control of ventilation. In: Cherniack, N.S., Widdicombe, J.G. (Eds.), Handbook of Physiology, Section 3, The Respiratory System: Control of Breathing, vol. 2. American Physiological Society, Bethesda, MD, pp. 793 – 813. Regan, M.J., Eager, E.J.I.I., 1966. Ventilatory responses to hypercpnia and hypoxia a normothermia and moderate hypothermia during constant depth anesthesia. Anesthesiology 27, 624–633. Smith, J.C., Greer, J.J., Liu, G., Feldman, J.L., 1990. Neural mechanisms generating respiratory pattern in mammalian brain stem-spinal cord in vitro I. Spatiotemporal patterns of motor and medullary neuron activity. J. Neurophysiol. 64, 1149– 1169. Smith, J.C., Funk, G.D., Johnson, S.M., Feldman, J.L., 1995. Cellular and synaptic mechanisms generating respiratory rhythm: Insights from in vitro and computational studies. In: Trouth, C.O., Millis, R.M., Kiwull-Scho¨ne, H.F., Schla¨fke, M.E. (Eds.), Ventral
Y. Fukuda / Neuroscience Research 38 (2000) 407–417 Brainstem Mechanisms and Control of Respiration and Blood Pressure. Lung Biology in Health and Disease, vol. 82. Marcel Dekker, New York, pp. 463–496. St John, W.M., 1996. Medullary regions for neurogenesis of gasping: noeud vital or noeuds vitals? J. Appl. Physiol. 81, 1865–1877. St John, W., Knuth, K.V., 1981. A characterization of the respiratory pattern of gasping. J. Appl. Physiol. 50, 984–993. St John, W., Bledsoe, T.A., Sokol, H.W., 1984. Identification of medullary loci critical for neurogenesis of gasping. J. Appl. Physiol. 56, 1008 – 1019. Suzue, T., 1984. Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J. Physiol. Lond. 354, 173 – 183.
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Thach, B.T., Jacobi, M.S., Gershan, W.M., 1991. Control of breathing during asphyxia and autoresuscitation. In: Haddad, G.G., Farber, J.P. (Eds.), Developmental Neurobiology of Breathing, Lung Biology in Health and Disease, vol. 53. Marcel Dekker, New York, pp. 681 – 699. Tschirgi, R.D., Gerard, R.W., 1947. The carotid-mandibular reflex in acute respiratory failure. Am. J. Physiol. 150, 358 – 364. Wang, W., Fung, M.-L., Darnall, R.A., St John, W.M., 1996. Characterizations and comparison of eupnoea and gasping in neonatal rats. J. Physiol. (Lond.) 490, 277 – 292. Yuan, S.-Z., Blennow, M., Runold, M., Lagercrantz, H., 1997. Effects of hyperglycemia on gasping and autoresuscitation in newborn rats. Biol. Neonate 72, 255 – 264.
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Respiratory neural activity responses to chemical stimuli in newborn rats: reversible transition from normal to ‘secondary’ rhythm during asphyxia and its implication for ‘respiratory like’ activity of isolated medullary preparation Yasuichiro Fukuda * Department of Physiology II, School of Medicine, Chiba Uni6ersity, 1 -8 -1 Inohana, Chuo-ku, Chiba City 260 -8670, Japan Received 26 June 2000; accepted 22 August 2000
Abstract To clarify a possible origin of ‘respiratory like’ rhythmic activities observed in in vitro brainstem preparation, the phrenic (Phr) and cranial nerve (XII or IX) inspiratory activities were analyzed in halothane-anesthetized, vagotomized and artificially ventilated newborn (2–6 days after birth) and young adult rats (30 – 50 days) during altered chemical stimuli and prolonged asphyxia at 25°C. The newborn rat showed regular rhythmic inspiratory discharges of short duration, and their responses to CO2 and hypoxia did not differ from those seen in adult rats. In the newborn rat the Phr and cranial nerve inspiratory discharges increased first, then respiratory frequency decreased and finally ceased completely for 1 – 2 min during asphyxia. Thereafter, ‘secondary’ rhythmic inspiratory activity emerged at a slower rate with decremental inspiratory discharge profile, which persisted for a period more than 40 min of asphyxia. A normal respiratory activity recovered after resumption of artificial ventilation. Though young adult rats exhibited similar sequential changes in respiratory activity during asphyxia, the ‘secondary’ rhythmic activity persisted for a period of several min only. The pattern of ‘secondary’ respiratory activity corresponded well with that of rhythmic activities seen in the isolated medullary block preparation of newborn rat. ‘Respiratory like’ activity found in isolated medullary preparations of newborn animals may arise from a mechanism that generates ‘secondary’ (or so called ‘gasping’ type) rhythmic inspiratory activity during prolonged asphyxia in in vivo preparations. © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Newborn rat; Phrenic and cranial nerve inspiratory activity; Normal respiratory rhythm; ‘Secondary’ respiratory rhythm; Asphyxia
1. Introduction Recent observations on the isolated brainstem (spinal cord) or medullary block/slice preparations obtained from newborn animals have provided essential evidence for a possible site(s) and cellular mechanisms underlying respiratory rhythm generation (Suzue, 1984; Smith et al., 1995; Onimaru et al., 1997). Onimaru and his group (Onimaru et al., 1987, 1988) found that the propriobulbar neurons showing pre-inspiratory dis-
* Tel.: +81-43-2262030; fax: + 81-43-2262034. E-mail address: [email protected] (Y. Fukuda).
charge pattern in the rostro-ventrolateral medulla involve primary rhythm generating mechanism. Smith et al. (1995) have hypothesized that the pre-Bo¨tzinger complex, a site within the ventrolateral medulla, contains the neuron population initiating a primary rhythm of respiration. These medullary neurons and their ‘respiratory like’ rhythmic activity found in in vitro preparations may also have functional role in the generation of normal respiratory rhythm in in vivo animals. Smith et al. (1990) identified rhythmic neural discharges of isolated brainstem spinal cord preparation with inspiratory discharges in vivo animals at a comparable experimental condition in the newborn rat. However, critical arguments have confronted such favorable interpretation. These include that output mo-
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toneurons of in vitro preparation display always ‘gasping’ type discharge and no one has reported normal respiratory activity pattern. There is a general agreement that substantial analog of in vitro ‘respiratory like’ activity should be found in in vivo preparation during altered chemical stimuli particularly during anoxia or asphyxia. Wang et al. (1996) comparing inspiratory discharge patterns between in vivo newborn rats during anoxia and in vitro preparations, stated that mechanism and site of generation of in vitro ‘gasping’ type activity differ from that of normal respiratory pattern. If this were the case (St John, 1996), two rhythm generators of different origins (‘gasping’ and normal types) would drive common outputs such as phrenic and cranial nerve motoneurons. However, validity of such two rhythm generator concept still requires further analysis of not only individual inspiratory burst profile but also changes in rhythmicity, temporal aspect of transition from normal to ‘gasping’ rhythms during anoxia and their reversibility. Concerning the respiratory rhythmicity during anoxia or asphyxia, ventilatory measurements in newborn animals by previous many studies (see review, Thach et al., 1991) have already established a sequence of changes in spontaneous ventilatory or air flow pattern in response to asphyxia (or anoxia): initial hyperpnea, primary apnea, hypoxic (or asphyxic) ‘gasping’ and secondary apnea. Asphyxic ‘gasping’ functions as an important autoresuscitation process for survival in newborn animals by which these animals show particular tolerance to anoxia and asphyxia (Thach et al., 1991). However, correlative neurophysiological analysis is required to confirm these findings. Perhaps asphyxic ‘gasping’ observed in in vivo neonates corresponds to respiratory like rhythmic activity found in in vitro preparation. These conflicting interpretations of the results depend presumably upon that respiratory neural activities of newborn rat have not been analyzed systematically and quantitatively at controlled levels of chemical stimuli or during asphyxia. The present study aims to analyze the phrenic and cranial nerve (XII or IX) inspiratory discharge responses to altered CO2 and hypoxic stimuli during eupnea and to prolonged asphyxia. Experiments were designed to observe sequential changes in phrenic and cranial nerve inspiratory activities during progression of asphyxia not only in the newborn but also in the young adult rats. The respiratory neural activities during asphyxia were further compared with rhythmic activity in in vitro medullary block preparations obtained from newborn rats. The results revealed a reversible shift of normal respiratory rhythmicity to a more slower ‘secondary’ (or ‘gasping’) respiratory rhythm during prolonged asphyxia. The latter coincided well with ‘respiratory like’ rhythmic activity found in isolated medullary block preparation.
2. Material and methods
2.1. General We used newborn rats (3–6 days: 5–10 g) and young adults rats (30–50 days: 110–160 g) of either sex. Following anesthetized with halothane (induction 2.0– 2.5%, maintenance during surgery 1.5–1.2%), animals were tracheotomized and artificially ventilated by a rodent respiratory at a rate of 80–100/min with a constant stroke volume (0.8–1.5 ml for newborns, 1.5– 2.5 ml for young adults) (Shinano Seisakusho, type 170, Tokyo) under subcutaneous administration of a muscle relaxant (Mioblock® (2 mg/ml), total 0.1 ml/body in the newborn and 0.8 ml/body in young adults). We sectioned bilateral vagus nerves at the mid cervical region. In the control condition body temperature was maintained at 37°C by a heating pad, a temperature regulator and a thermistor probe (tip diameter 1 mm) placed in the rectum. Intra-tracheal gas was continuously sampled to measure fractional concentration of CO2 and O2 (FCO2, FO2) by a gas analyzer (25 ml/min, NEC-Sanei Instruments, type 1H26, Tokyo). The tracheal peak FCO2 and the inspiratory FO2 values were maintained at about 0.07–0.08 and above 0.50, respectively, in the control condition by adjusting the amount of CO2 and O2 gas flowing into the inspiratory line. The phrenic and hypoglossal (or glossopharyngeal) (XII or IX) nerves were separated from surrounding tissues and dissected for recording inspiratory activity by a bipolar Pt–Ir electrode in liquid paraffin/mineral oil mixture. Although the onset and fading of respiratory rhythmic activity are best detected by inspiratory discharge of IX nerve (Fukuda et al., 1995), recording of IX nerve activity was sometimes difficult in in vivo and in vitro newborn rats. We, therefore, analyzed inspiratory burst duration of Phr and XII nerves. Original neural activity and its leaky integrated wave were recorded on a chart recorder and were stored on magnetic tape for later analysis. We also recorded ECG as an index of cardiac function.
2.2. Reduction of body temperature To compare rhythmic activities between in vivo whole animal and in vitro preparation at the same temperature condition, the rectal temperature was decreased from 37 to 25°C by cooling the body surface as described previously (Maruyama and Fukuda, 2000). This was because in vitro brainstem (-spinal cord or medullary block) preparations have been examined at lower temperature (24–27°C) to maintain viability of isolated brain tissue. Due to significant temperature effects on anesthesia (Pavlin and Hornbein, 1986) the depth of anesthesia at 25°C was maintained at an almost similar level of ‘depth’ with that in control 37°C
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by decreasing inspiratory halothane concentration from 1.1% in normothermia to 0.4 – 0.5% during hypothermia (25°C) (Maruyama and Fukuda, 2000). This adjustment of halothane concentration was critically important for the analysis of respiratory control mechanism in response to chemical stimuli during induced hypothermia (Regan and Eager, 1966).
2.3. Effect of changing chemical stimuli and asphyxia The effect of CO2, hypoxia and asphyxia on Phr and XII (or IX) inspiratory discharges was examined at 25°C. The level of CO2 stimulus was altered by changing the amount of CO2 injected into the inspiratory line with monitoring peak tracheal FO2 value. We also tested effects of step wise reduction in inspiratory FO2 (FIO2) from above 0.50 down to 0.05. Effects of asphyxia were examined as follows. After recording respiratory nerve activity in the control hyperoxic (FIO2 \0.50) and slightly hypercapnic condition (peak FCO2 =0.07 – 0.08%) at 25°C, artificial ventilation was ceased for more than 50 min in the newborn and about 10 min in the young adult rats. Changes in Phr and XII (or IX) inspiratory activities were continuously recorded during cessation of artificial ventilation and during recovery after resumption of artificial ventilation.
2.4. Isolated medullary block preparation of newborn rat Seven newborn rats (2 – 6 days) of either sex were deeply anesthetized with halothane. The lower brainstem was quickly isolated and placed immediately in a cold mock CSF (4°C). A transverse section of medullary region from a level of obex and the lower edge of trapezoid body (thickness 1.5 mm) was made manually with using a steel razor blade in cold mock CSF (4°C), and the tissue block was placed in a continuously flowing mock CSF in an incubation chamber at 25°C. The mock CSF had following composition in mM: NaCl 135, KCl 4.0, CaCl2 1.3, MgSO4 0.6, NaH2PO4 0.6, NaHCO3 24 and glucose 20, and the solution had pH of 7.40 when equilibrated with 95%O2 – 5%CO2 gas mixture. The ‘respiratory like’ activity was monitored from the XII rootlet with a suction electrode (tip internal diameter 0.05 mm), and a unit activity was simultaneously recorded from the ventral medullary region by a glass capillary microelectrode (tip diameter 2 mm). We compared the rate of rhythmic activity and individual (inspiratory) burst pattern between in vivo preparation during asphyxia and in vitro preparations.
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2.5. Data analysis Normal respiratory frequency, Phr and XII inspiratory duration was compared between young adult and newborn rats during hyperoxic breathing at 37 and 25°C. The rate of ‘secondary’ rhythmic activity and inspiratory bursts duration during asphyxia were compared with that obtained in hyperoxia at 25°C. Furthermore, the rate of rhythmic activity was compared quantitatively between in vivo whole animal preparations during prolonged asphyxia and in vitro medullary block preparations. We considered statistically significant difference (PB 0.05) of compared data by ANOVA and Bonferroni correction of t-test.
3. Results
3.1. Comparison of phrenic and cranial ner6e inspiratory acti6ities between newborn and young adult rats Fig. 1 compares inspiratory activities of the Phr and cranial nerves (IX or XII) between newborn and young adult rats at control condition (hyperoxia, peak tracheal FCO2 0.07–0.08, 37°C). Although the rate of rhythm (respiratory frequency) did not differ between newborn and young adult rats, newborn rats showed short duration of Phr and XII burst (Table 1, Fig. 1). With reduction in body temperature from 37 to 25°C, respiratory frequency decreased and the duration of the Phr and XII nerve inspiratory bursts prolonged significantly in both young adult and newborn rats (Table 1, Fig. 2). In the newborn rat, each Phr or cranial nerve inspiratory burst often consisted of a train of several short bursts. The newborn rat showed significantly slower respiratory frequency and shorter duration of Phr and XII inspiratory bursts than the young adult rat at 25°C in hyperoxia (Table 1).
3.2. Inspiratory acti6ity response to chemical stimuli in the newborn rat The newborn rats showed almost similar responses of Phr and XII inspiratory discharge to changing the level of chemical stimuli as usually seen in normothermic adult rats. Respiatory frequency and inspiratory discharges of newborn rats increased with elevating tracheal FCO2, and respiratory rhythmicity ceased during hypocapnia (hypocapnic apnea) (Fig. 3). At a higher peak FCO2 (above 0.08), however, respiratory frequency rather decreased (Fig. 3D). Effects of decreasing FIO2 are shown in Fig. 4. Respiratory frequency and inspiratory discharges increased as decreasing FIO2 from 0.40 to 0.08 accompanying with rapid onset of Phr activity. Furthermore, the onset of cranial nerve activity became
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Fig. 1. Respiratory neural activity of halothane anesthetized, vagotomized and artificially ventilated young adult (A) and newborn rats (B,C) at 37°C in hyperoxia. (A) Young adult rat (30 days); (B) newborn rat (3 days); (C) newborn rat (2 days). Paw, airway pressure; FCO2, fractional concentration of CO2 in tracheal gas; Phr, phrenic nerve activity; Phr, integrated phrenic nerve activity; IX, glossopharyngeal nerve activity; integrated glossopharyngeal nerve activity; XII, hypoglossal nerve activity; XII, integrated hypoglossal nerve activity. These abbreviations are the same in the following figures.
Table 1 Respiratory neural activity of newborn and young adult rat at 37°C (hyperoxia, normal rhythm), 25°C (hyperoxia, normal rhythm), and during asphyxia (‘secondary’ rhythm) in in vivo whole animal preparations and in isolated medullary block preparations (newborn rat only) Condition
n
f (min−1)
Newborn rats (in 6i6o) 37°C, hyperoxia (normal rhythm) 25°C, hyperoxia (normal rhythm) 25°C, asphyxia (‘secondary’ rhythm)
(8) (8) (8)
31.8 95.8 16.3 93.1* 5.6 91.0 c
Newborn rats (isolated medullary preparation)
(7)
6.5 90.8
Young adult rats (in 6i6o) 37°C, hyperoxia (normal rhythm) 25°C, hyperoxia (normal rhythm) 25°C, asphyxia (‘secondary’ rhythm)
(7) (7) (7)
31.3 95.0 26.0 93.7* 18.3 93.0 c ,++
Phr duration (ms)
188 9 64 526 9189* 320 9 122 c
XII duration (ms)
394 9 138 822 9312* 285 987 c 464 956+
669 9141 1092 9 137* 407 9152 c
910 9 95 1448 9225* 289 9 84 c
* Statistically significant difference (PB0.05) between 37 and 25°C in hyperoxic conditions. c Statistically significant difference (PB0.05) between hyperoxic and asphyxic conditions at 25°C. + Statistically significant difference (PB0.05) between in vivo asphyxic and in vitro isolated preparations at 25°C (newborn rat only). ++ Statistically significant difference (PB0.05) between newborn and young adult rats at 25°C during asphyxia.
Fig. 2. Effect of decreasing body temperature from 37 (A) to 25°C (B) on respiratory nerve activity in newborn rat (5 days).
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Fig. 3. Effect of changing CO2 stimulus on respiratory nerve activity in newborn rat (4 days) during hyperoxia (FIO2 \0.50) at 25°C.
Fig. 4. Effect of changing FIO2 on respiratory nerve activities in newborn rat (2 days) during isocapnia (peak FCO2, 0.06) at 25°C.
Fig. 5. Antagonistic effect of hypocapnic (peak FCO2 0.04) inhibition and hypoxic stimulation (FIO2 0.08) on respiratory nerve activities in newborn rat (4 days) at 25°C.
earlier and the duration of inspiratory bursts prolonged greatly. However, respiratory frequency determined by rhythmic Phr activity decreased with further reduction in FIO2 to 0.05 (Fig. 4). Some cranial nerve bursts did not accompany with synchronized Phr burst. As shown in Fig. 5, when respiratory rhythmicity ceased during
hypocapnia (peak FCO2 0.03–0.04), the reduction in FIO2 (0.08) induced regeneration of rhythmicity and augmented the inspiratory discharges. Such counteracting effect of hypoxia and hypocapnia was completely similar to those usually seen in adult rats at normothermia (figure not shown).
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Fig. 6. Effect of prolonged asphyxia on respiratory nerve activities in newborn rat (3 days) at 25°C. (A)–(D) were obtained from the same rat before and during asphyxia. Asphyxia was produced by cessation of artificial ventilation. (B) was obtained at 1 min after (A), (C) was 10 s after (B), and (D) was 30 min after (C).
3.3. Inspiratory acti6ity during asphyxia and reco6ery 3.3.1. Newborn rats Following cessation of artificial ventilation, respiratory frequency and the magnitude of integrated Phr and cranial nerve activity increased gradually, then fre-
quency decreased with prolongation of expiratory time, and finally respiratory rhythm expired completely for 1–2 min (primary apnea, Fig. 6). Individual inspiratory burst pattern of Phr and XII nerves changed from normal augmenting to abrupt onset with long lasting decremental pattern just before cessation of normal rhythm (Fig. 7). The onset of cranial nerve inspiratory activity became much earlier initially and then became almost simultaneous before cessation of rhythm. Normally within several minutes of apnea, a new rhythmic activity appeared first in the cranial nerves which was later followed by the appearance of Phr inspiratory activity. We defined such new rhythm as ‘secondary’ respiratory rhythm. At this initial stage, the onset of cranial nerve inspiratory activity preceded the start of Phr discharge as seen before asphyxiation. The Phr inspiratory activity showed an abrupt onset and decremental decay, which was indistinguishable from those seen before cessation of normal rhythm (compare ((2), (3)) and ((5), (6)) in Fig. 7). During a later steady state condition, the onset of inspiratory activity became almost simultaneous in both the Phr and cranial nerves (Fig. 7), and each inspiratory activity showed abrupt onset with decremental firing pattern. These synchronized rhythmic Phr and cranial nerve inspiratory activities persisted for a period more than 40 min of asphyxia. At that time there was neither QRS wave in the ECG tracing (figure not shown) nor visible pulsation in the carotid artery suggesting almost total arrest of arterial circulation. As shown in Table 1, the rate of ‘secondary’ rhythmic activity at steady state condition (20–40 min asphyxia) was significantly slower than that of normal rhythm observed during hyperoxic control condition. There was no significant difference in the duration of Phr and XII inspiratory burst duration between normal and ‘secondary’ rhythmic activities during asphyxia. The rate of secondary rhythm and amplitude of integrated Phr or XII inspiratory discharges decreased gradually and finally be came undetectable (final apnea) at 60–80 min asphyxia.
Fig. 7. Shape of individual inspiratory discharge profile at various time before and during asphyxia indicated by numbers at the top of each trace in Fig. 6.
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Fig. 8. Recovery of respiratory activity after prolonged asphyxia in newborn rat (3 days) at 25°C. (A) – (C) were obtained from the same animal. (A) Before asphyxia; (B) during asphyxia and recovery; (C) 5 min after (B). Individual inspiratory burst profile at various time indicated by number and allow is shown in higher chart speed recording in lower trace.
Following resumption of artificial ventilation at 40– 50 min of asphyxia, ECG activity (QRS wave) and carotid pulsation recovered first (figure not shown). The ‘secondary’ rhythmic activity ceased for a while (1–3 min) (Fig. 8B), thereafter respiratory rhythm recovered initially at a slow rate and later became faster and finally returned to pre-asphyxic level (Fig. 8C). Shape of individual inspiratory bursts recovered also. When asphyxia test was repeated, normal rhythm shifted smoothly to secondary rhythm without interruption by clear primary apnea period. Normal or ‘secondary’ respiratory rhythmic activities did not recover with resumption of artificial ventilation when duration of asphyxia exceeded 90 min.
3.3.2. Young adult rats Typical example of changes in respiratory activity during asphyxia in the young adult rat is presented in Fig. 9. After cessation of artificial ventilation, inspiratory discharges of Phr and cranial nerves increased initially, then became tonic and finally ceased completely for 2–3 min. ‘Secondary’ rhythmic activity appeared first in the Phr nerve which was later followed by small synchronized cranial nerve inspiratory discharges. The pattern of’secondary’ rhythmic activity during asphyxia differed from that in hyperoxic pre-asphyxic condition (Fig. 9 and Table 1). The respiratory frequency of asphyxic ‘secondary’ rhythm was significantly lower than that during hyperoxia. The Phr inspiratory activity during ‘secondary’ rhythm was gradual in onset reaching peak with gradual decline. These ‘secondary’ respiratory activities became slower and smaller and terminated within several min (Fig. 9), which was associated with circulatory arrest. Neither
respiratory rhythmic activities nor ECG activities recovered even with resumption of artificial ventilation at 5–6 min asphyxia (Fig. 10).
3.4. Pattern of rhythmic acti6ity in isolated medullary block preparation of newborn rat Rhythmic mass discharge was consistently recorded from the XII nerve root in the medullary block preparation of newborn rat. The rhythmic XII activity fired synchronously with burst of rhythmic unit activity recorded from rostro-ventral medullary region and its vicinity (Fig. 11). There was no significant difference between the rate of rhythmic activity in isolated medullary preparations and that of ‘secondary’ respiratory activity in in vivo preparation during asphyxia (Table 1). Furthermore, , decremental discharge pattern of the XII nerve activity in isolated preparation was almost analogous to that of the XII inspiratory discharge of in vivo new born rat during steady state asphyxic condition (Fig. 11).
4. Discussion We have recorded successively respiratory nerve discharges and their responses to chemical stimuli in the newborn rat. A high respiratory frequency and short duration of Phr and XII (or IX) inspiratory bursts characterized a feature of respiratory neural pattern of newborn rat during hyperoxic control condition. The newborn rats showed almost similar respiratory nerve activity responses to CO2 and hypoxia as seen in normal adult rat. Therefore chemical control mechanisms
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Fig. 11. Comparison of asphyxic ‘secondary’ respiratory activity of in vivo rat (4 days) (A) and rhythmic activity observed in in vitro medullary block preparation (rostro-ventral medullary region) of newborn rat (3 days) (B) at 25°C. (A) and (B) were obtained from different animals.
of respiratory system have, at least qualitatively, well developed at 2–6 days after birth. The present experiments demonstrated following three results. First, pattern of respiratory motoneuron discharges shifted from normal to a slower ‘secondary’ rhythmic type during prolonged asphyxia, and normal respiratory pattern recovered after resumption of artificial ventilation in newborn rats. Second, young adult rat exhibited also a similar ‘secondary’ respiratory activity during asphyxia but for a short period only. Third, pattern of’secondary’ respiratory activity was indistinguishable from rhythmic activity found in in vitro medullary block preparation of newborn rat. Fig. 9. Effect of prolonged asphyxia on respiratory nerve activities in young adult rat (35 days) at 25°C. (A)–(D) were obtained from the same animal. (A) and (B) were continuous recordings, (C) was obtained 6 min after (B). Shape of individual inspiratory burst profile at time indicated by number is shown in (D) with higher chart speed recording.
4.1. Respiratory pattern change during asphyxia Prolonged asphyxia in the newborn rat initiated a sequence of change in respiratory activity, i.e., augmentation of Phr and XII (or IX) inspiratory activities, cessation of rhythmicity (primary apnea), generation of
Fig. 10. Effect of asphyxia on respiratory nerve activities in young adult rat (30 days) at 25°C. (A) and (B) were obtained from the same animal. (B) was 1 min after (A). Note that secondary rhythmic activity disappeared and normal rhythmic activity did not recover even after resumption of artificial ventilation.
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‘secondary’ rhythm and final apnea. These systematic alterations correlated well with previous observations on ventilatory pattern change during asphyxia (hyperpnea, primary apnea, hypoxic ‘gasping’ and secondary apnea) (Thach et al., 1991). Although the term ‘gasp’ has been used in reference to the last respiratory pattern appearing prior to death in the course of asphyxiation, ‘gasp’ has now been viewed as coordinated and functional respiratory activity (Thach et al., 1991). We, therefore, used ‘secondary’ respiratory activity instead of ‘gasp’. In neurophysiological studies ‘gasp’ type activity during hypoxia is characterized by strong inspiratory activity with abrupt onset and gradual decay (decremental pattern) (St John, 1996). However, as seen in Fig. 4, Phr inspiratory discharge profile became abrupt in onset and with gradual decay during graded reduction in FIO2 (Fig. 4, FIO2 0.05) and also during an early phase of asphyxia even during continuation of normal respiratory rhythm (Fig. 7). Discharge profile of individual inspiratory bursts alone, i.e. decremental discharge pattern, thus, represents neither alterations of rhythm generation nor specific feature of ‘gasping’ type respiratory rhythm, but shows the presence of strong hypoxic (and hypercapnic) drive to inspiratory output motoneurons. Normal and ‘secondary’ respiratory rhythm differed significantly in their rate of rhythmicity (Table 1), which suggested a possibility that two rhythm generators of different origin with different inherent rhythmicity could drive common Phr and XII (or IX) motoneurons. Because normal respiratory activity recovered after resumption of artificial ventilation, the mechanism generating normal respiratory rhythm had not been deteriorated but rather suppressed functionally during prolonged asphyxia. A reversible hypoxic inhibition of respiratory activity has been well known and seen particularly in newborn or young animals (Haddad et al., 1982; Mortola, 1993; Fukuda, 1992). Accumulation of inhibitory substances such as adenosine, opioids, GABA accounts for occurrence of hypoxic respiratory inhibition (Neubauer et al., 1990), particularly of respiratory frequency or rhythm generation (Fukuda, 1991), which leads to the cessation of normal rhythm (primary apnea). A simple explanation is that following hypoxic inhibition of normal rhythm generation a separate mechanism of initiating ‘secondary’ rhythm becomes active to drive common inspiratory outputs, i.e. Phr or XII motoneurons. St John and Knuth (1981), St John (1996) have hypothesized separate sites of ‘gasp’ and normal (eupneic) respiratory medullary centers. This hypothesis necessarily requires an assumption that mechanism of ‘secondary’ (or ‘gasping’) rhythm is inactive or inhibited in hyperoxic condition. Another possibility may include that the rate of rhythmicity of normal rhythm generator in the medulla is changed by deprivation of inputs from other neural structures during prolonged asphyxia (see review,
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Thach et al., 1991). This case does not require a separate ‘gasp’ center in the medulla oblongata. Further analyses are necessary on transition mechanism(s) from normal to ‘secondary’ rhythms and it neuroanatomical correlates. In the present study we did not analyze correlation between circulatory and respiratory alterations during asphyxia. Thach et al. (1991) indicated possible importance of cardiovascular function for maintenance of gasping. However, the fact that ‘secondary’ respiratory activity during asphyxic condition was seen under almost total circulatory arrest suggested that generation of ‘secondary’ respiratory activity might have appeared without obvious dependency on intact medullary blood supply.
4.2. Comparison between newborn and young adult rats Young adult rats showed similar sequential change in respiratory activity during asphyxia as in newborn rats, i.e. respiratory stimulation followed by suppression leading to apnea, generation of ‘secondary’ respiratory rhythm and final apnea. The average rate of ‘secondary’ rhythm was significantly slower than that during control condition as in newborn rats. However, the duration of ‘secondary’ rhythm was much shorter than that of newborn rats (10 min vs. more than 40 min). Yuan et al. (1997) also described that 1-day-old rats gasped much longer than the 8-day-old rats. Much shorter period of displaying asphyxic ‘secondary’ respiratory rhythm and a lack of recovery of respiratory rhythm after resumption of artificial ventilation in the young adult rat may be due to irreversible damages of neural mechanism generating respiratory rhythm during prolonged asphyxia. Jacobi and Thach (1989) studied the effect of maturation on the ability of autoresuscitation by ‘gasping’ in mice and found that decrease in survival in older mice. Tolerance to hypoxia decreases gradually during early developmental phase after the birth (Duffy et al., 1974). Altered vulnerability of neural tissues to anoxia depends upon changes in metabolic states, i.e. transition from predominance of anaerobic to aerobic during development (see review, Nioka and Chance, 1991). The neuronal mechanism initiating ‘gasp’ may depend largely upon anaerobic metabolism (Tschirgi and Gerard, 1947). Yuan et al. (1997) found that hyperglycemia prolongs the duration of anoxic ‘gasping’ period in the newborn rats of 8 days age suggesting glycolytic energy supply for generation of ‘gasping’ type respiratory activity. The differences in asphyxic tolerance due to postnatal age involve various O2 and metabolic related factors such as hemoglobin’s O2 affinity, O2 consumption, O2 availability, glycogen storage and/or mitochondrial capability (Nioka and Chance, 1991). In the young adult rats phrenic inspiratory bursts during ‘secondary’ rhythm was gradual in onset reach-
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ing peak followed by gradual decay, which contrasted to abrupt onset and decremental decay in the newborn rat. This finding again suggested the pattern of individual inspiratory bursts may not characterize particular feature of asphyxic ‘secondary’ (or ‘gasping’) respiratory activity.
4.3. Comparison of rhythmic (respiratory) acti6ity between in 6i6o and in 6itro preparations Despite completely different experimental approaches, the rate and shape of rhythmic activity did not differ between in vivo preparation during asphyxia and in in vitro preparations. A plausible explanation may be that anoxia tolerant mechanism of generating ‘secondary’ (or ‘gasping’ type) respiratory rhythmicity had been still active and functioned even in in vitro medullary preparations. Suzue (1984), in his pioneer study, already suggested the rhythmic pattern of in vitro brain stem spinal cord preparation may be ‘gasping’. Absence of ‘normal’ respiratory like rhythmic activity in in vitro preparation is due either to a lack of enough tissue oxygenation to activate its generating mechanism even by superfusion with hyperoxic mock CSF or to irreversible tissue damages in isolated condition. However, in vitro rhythmic activities were recorded from medullary block preparations superfused with hyperoxic and normocapnic solution while ‘secondary’ respiratory activity was seen during as phyxia, i.e. hypoxic and hypercapnic condition. Although local PO2 and pH values in deeper medullary tissues have been shown to be lower in brain-stem spinal cord preparations (Okada et al., 1993), these were not measured in the present experiment. Tissue PCO2 and PO2 of isolated medullary block preparations should be measured in future studies. Effects of hypoxia and/or hypercapnia may also be examined in isolated medullary preparation. Recent observation on the isolated medullary slice of mice showed that normal respiratory, sighs or gasping type activities are all recorded from a single medullary area, preBo¨tzinger region under application of high KCl (8 mM) in superfusing solution and with application of synaptic inhibitors (Lieske et al., 2000). A comparison between in vivo and in vitro preparations is required in mice. Further experiments are also necessary on in vivo newborn animals in which the medulla oblongata is isolated totally from surrounding tissues to remove influences from higher centers or from peripheral afferents including vagal and carotid chemoreceptor afferents. Although St John et al. (1984) showed that gasp-like breathing in normoxic and brain stem lesioned cats is little affected by carotid chemoreceptor afferents, roles of peripheral chemoreceptor afferents in the generation of asphyxic ‘secondary’ (or ‘gasping’) rhythmic activity have not been elucidated (Thach et al., 1991).
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