The effects of lowering body temperature (Tb) on metabolic rate, ventilation, and the strength of the Hering-Breüer expiratory promoting reflex (HB reflex; determined from an inhibitory ratio calculated from volumetric measurements of the respiratory rhythm) were examined in 18-day-old ectothermic pouch young of the tammar wallaby during normoxia or hypoxia (10% O2). Hypoxia and hypothermia, either singularly or combined, depressed metabolic rate. At all Tb, the hypoxic hyperventilation was associated with a significant hyperpnea. At pouch Tb (36.5°C) during normoxia, inflation of the lungs with -5 or -10 cmH2O extrathoracic pressure induced a significant HB reflex. Exposure to cold reduced the strength of the reflex, almost abolishing it at 28°C. For Tb above 28°C, the reflex in hypoxia was always less than the corresponding normoxic value. Taken in context with the changes in metabolic state that occurred, these data in the ectothermic marsupial newborn suggest that the decline in the HB reflex during moderate hypothermia is the result of a direct effect of Tb on vagal mechanisms rather than a temperature-driven decline in metabolic rate that should have acted to strengthen the HB reflex. Therefore, it seems that inputs inhibitory to breathing are more negatively affected during cold than those inputs that are excitatory.
- metabolic rate
- breathing pattern
when the airways are occluded or lung volume is maintained elevated above functional residual capacity (FRC), the tonic discharge of pulmonary slowly adapting stretch receptors mediates an inhibition to breathe (41). This is known as the Hering-Breüer reflex (HB reflex). The inhibition results in a prolonged period of apnea that persists for a time course that depends, in part, on the elevation of lung volume above FRC (17) and the accumulation time of the chemical drive to breathe. The chemical drive, which is of metabolic origin (hypoxia as a result of the rate of O2 consumption, hypercapnia as a result of the rate of CO2 production), builds during the maintained apnea and eventually offsets the inhibition (43). The strength of the HB reflex is determined by the relative efficacies of the various parameters within the reflex loop; among these perhaps the most important are chemosensitivity and metabolic rate (25, 26, 43).
Changes in metabolic state influence the strength of the HB reflex. For example, the reflex is weaker when metabolic rate is increased, such as occurs with thermogenesis during cold exposure, because the chemical drive to breathe builds more rapidly, resulting in an earlier offset of the vagal inhibition (25, 26). Imposed hypoxia also weakens the HB reflex because of the increased chemical drive (3). In newborns, however, the weakening of the HB reflex with inspired hypoxia may be opposed by the decreased endogenous drive that is associated with a hypoxia-induced hypometabolism (27, 29). Furthermore, in newborn mammals, vagal ventilatory inhibition is not very sensitive to the chemical drive to breathe (22) and chemosensitivity is characteristically low (25-27). In general, age-related differences in the strength of the HB reflex exist because of the maturational differences in peripheral chemoreception (22).
Chemoreceptors and slowly adapting stretch receptors are sensitive to changes in temperature (23, 38) and, therefore, body temperature (Tb) would be expected to be an important determinant of the strength of the HB reflex. In the newborn rat, ambient temperature (Ta) would appear to be the important variable determining Tb, as the presence of a metabolic response to cold in normoxia (i.e., thermogenesis) or its absence in hypoxia makes no appreciable difference to the effect of Ta on Tb (28). On the other hand, the fact that at various Ta, Tb was similar between normoxia and hypoxia, whereas the strength of the HB reflex, determined from volumetric data, was very different (25, 26), suggests that in the newborn rat the effects of cooling on the strength of the HB reflex are not attributable to Tb per se, but to the corresponding changes in metabolic rate. However, to what extent the lowered Tb in normoxia would act via the Q10 effect (change in a rate process over a 10°C temperature range) to limit the changes in metabolic rate and hence the strength of the HB reflex is confounded by the induction of thermogenesis in normoxia. Furthermore, on exposure to hypoxia, the thermogenic component of metabolic rate becomes attenuated and the temperature effects on the strength of the HB reflex were instead confounded by the induced hypoxic drive as well as the effect of temperature on the peripheral chemoreceptors.
Marsupial pouch young (joey), particularly the macropods (kangaroos, wallabies, and potoroids), are incapable of thermogenesis for a large part of their development and rely on being raised within a thermally stable pouch (34). The ectothermic nature of the joey provides an opportunity to better distinguish cause and effect of changes in metabolic rate and Tb on the HB reflex. Therefore, we investigated the effects of altered Tb and hypoxia on the strength of the HB reflex determined from an inhibitory ratio calculated from volumetric measurements of the respiratory rhythm in 18-day-old tammar wallabies, Macropus eugenii, after first establishing that this age group was capable of a marked HB reflex in response to lung inflation.
MATERIALS AND METHODS
The strength of the HB reflex was measured at pouch temperature (normothermia = 36.5°C) in young tammar wallabies from birth to 27 days of age. The effects of temperature and/or hypoxia (10% O2) on the HB reflex, ventilation, and metabolic rate were subsequently examined in 18-day-old joeys. Animals were sourced from an outdoor captive-breeding colony maintained by the laboratory. Females had their pouch inspected on a daily basis to ascertain the day of birth (day 0). On the day of the experiment, the pouch was opened and the joey was carefully removed from the teat and immediately weighed to the nearest 1 mg. All experiments were conducted following approval from the Animal Ethics Committee of the institution.
Set up. A detailed account of the system used to measure the rates of O2 consumption and CO2 production (V̇o2 and V̇co2) and ventilation (V̇e) has been previously described (20). In brief, the apparatus was constructed from a water-jacketed chamber ∼50 ml in volume connected to a temperature-controlled water bath. A small mask, made from a short length of polyethylene tubing, was sealed to the face of the animal with a nontoxic polyether medical compound (Impregum F, Polyether Impression Material, ESPE). The other end of the tube was then inserted through a hole in the middle of a thin rubber stopper. In turn, the stopper was inserted midway along the length of the chamber, effectively dividing it into two separate compartments: one compartment communicated with the airways of the animal and the other enclosed its body. The atmosphere was maintained saturated by placing a moist gauze swab in each compartment.
HB reflex. A pneumotach (constructed according to Ref. 30) was inserted directly into the open end of the mask (Fig. 1A). Inspiratory and expiratory airflow was detected using a sensitive pressure transducer (±5 cmH2 2O. The inflation pressures were applied in a random order at 1-min intervals and each was repeated five times in each experimental condition. On each occasion, the pressure was maintained until the animal made its next inspiratory effort. The HB reflex was quantified by the inhibitory ratio (IR) and was calculated from the expiratory time during lung inflation (TeINF) divided by the average expiratory time (Te) of the five breaths immediately before the application of the vacuum: IREXP = TeINF/Te (Fig. 1B). An IREXP value significantly greater than one implies the HB reflex was induced. Furthermore, because the HB reflex is also known to be inspiratory inhibiting, as well as expiratory promoting, an inhibitory ratio was also calculated from the inspiratory time of the first breath that followed TeINF while the lung remained inflated (TiINF) divided by the average inspiratory time (Ti) of the five breaths immediately before the application of the vacuum: IRINS = TiINF/Ti (Fig. 1B).
Metabolic rate and ventilation. For the measurement of metabolic rate, the pneumotach was removed from the mask and the compartment communicating with the airways was sealed by inserting a rubber stopper. Incurrent and excurrent tubes connected to a roller pump enabled the compartment to be continuously flushed with gas. The compartment was sealed for ∼2-3 min by turning three-way taps located at the inlet and outlet of the compartment. The taps were then switched back and the compartment was flushed, forcing the gas through a small column of Drierite (Drierite, Hammond Drierite) before being analyzed for fractional concentrations of O2 and CO2o2) and CO2 production (V̇co2) were calculated from the time integral of the gas concentration curve, multiplied by the flow and the reciprocal of the time during which the compartment was sealed. These equations do not take into consideration the respiratory exchange ratio (11).2 consumption (V̇
Ventilation was recorded during the 2- to 3-min period that the compartment was sealed. A sensitive pressure transducer (model PT5, Grass Instruments, ±5 cmH2O) was connected to the compartment that communicated with the airways, and pressure oscillations that were associated with breathing were recorded, volume being calibrated by injection of 10 μl of room air. On each occasion an average of 50 consecutive breaths were analyzed as previously described (20; see Fig. 1B, inset) for inspiratory (Ti) and expiratory times (Te), total breath duration (TTOT = Ti+Te), postinspiratory pause (Tp) (note that Tp is fractional to Te because it represents the passive-static component of Te, achieved with closed glottis), dynamic expiratory time (T′e, i.e., lung deflation), tidal volume (Vt), breathing frequency (fR = 60/TTOT) and ventilation (V̇e = Vt · fR).
Protocols. The strength of the HB reflex was measured in tammar joeys at 6 h, 1, 7, 18, and 27 days after birth under pouch conditions (36.5°C, normoxia). Tammar wallabies aged 18 days were the youngest age group that showed a marked response to lung inflation (see Fig. 2).
A total of 17 animals aged 18 ± 1 day (mean ± 1 SE) was first measured under control conditions (36.5°C, normoxia) and then either at 1) 28 and 20°C, followed by hypoxia at 20°C (n = 6); 2) exposed to hypoxia and measured at 36.5, 28, and 20°C (n = 6); or 3) measured at 32°C followed by hypoxia at 32 and 20°C (n = 5). The three protocols were adopted because exposure to each test temperature lasted between 20 and 30 min, and the time required for each condition would prevent an individual animal from proceeding in a random order through all conditions. Measurements commenced after stabilization of the water bath, usually within 8-10 min. In a preliminary experiment with animals of this age it was found that Tb equilibrated within ±0.5°C of chamber temperature in 10 min.
Statistical analysis. Data are presented as means ± 1 SE. Comparisons between each experimental group were performed by ANOVA and, if appropriate, taking into consideration repeated measures, followed by post hoc Bonferroni limitation modified t-tests between conditions of interest. In all cases, significance was considered at P < 0.05.
HB reflex at various ages. In tammar joeys 6 h after birth, inflation of the lungs with -10 cmH2O failed to elicit an HB reflex, quantified by IREXP (Fig. 2). A weak, although insignificant, reflex was present by day 1, but became more pronounced with increasing age. There was no discernable difference in the reflex between 18 and 27 days of age.
Effects of Tb and hypoxia on metabolic rate and ventilation. Values for breathing pattern, ventilation, and metabolic rate under pouch conditions for 18-day-old joeys are presented in Table 1. The breathing pattern was characterized by a long postinspiratory pause, typically observed in marsupial joeys, and is achieved through closure of the glottis at end of inspiration (8, 10). In normoxia at 18 days of age, a decrease in Tb resulted in a decrease in metabolic rate, with Q10 values similar to those expected for an ectotherm (Fig. 3). At all temperatures, V̇e remained tightly coupled to metabolic rate such that the convective requirement (V̇e/metabolic rate) was maintained (Fig. 4). Adjustments to V̇e with decreasing temperature were achieved mainly through changes in fR with Vt remaining unchanged, apart from 20°C. The decrease in fR (i.e., prolongation of TTOT) that occurred with decreasing temperature was largely the result of a lengthening in Tp, with Ti and T′e remaining constant, except at 20°C, where the breathing pattern became irregular and there was a marked increase in Ti and Tp (Fig. 5). Exposure to hypoxia at all Tbs resulted in a similar degree of hyperventilation (Fig. 4) that was achieved both by a hypometabolism (-29%) and a hyperpnea (+27%). At 36.5 and 32°C, the hyperpnea was the result of an increase in Vt (+51%) and slight decrease in fR (-13%), whereas at 28 and 20°C, it was predominately driven by an increase in fR (+20%).
Effects of Tb and hypoxia on the HB reflex. During normoxia, the IREXP was independent of stimulus intensity for a given temperature and weakened with decreasing temperature, being nearly abolished at 28 and 20°C (Fig. 6A). At temperatures where the reflex occurred (36.5 and 32°C), hypoxia weakened the IREXP elicited with an inflation pressure of -10 cmH2O and inhibited the reflex at -5 cmH2O (Fig. 6A). A small IRINS existed that was independent of stimulus intensity, level of oxygen, and Tb (Fig. 6B).
The TeINF-Te relationship (Fig. 7) reveals that at temperatures >32°C TeINF is independent of Te. Whereas IREXP is seemingly abolished <32°C, prolongation of Te at low temperatures is accompanied by a proportional change in TeINF; however, TeINF remained longer than Te, as evidenced by most data points lying above the isolateral line.
Effects of metabolic rate on the HB reflex. At temperatures where the IREXP was discernable, a reduction in metabolic rate, whether it was caused by temperature (hypothermia) or hypoxia (hypoxic-induced hypometabolism), resulted in a decrease in the strength of the reflex (Fig. 8).
In the 18-day-old tammar wallaby joey inflation of the lungs under pouch conditions (36.5°C, normoxia) resulted in a substantial prolongation of expiration until the next inspiration. This response to lung inflation is characteristic of the HB reflex, which is commonly observed in many newborn mammals (16, 33, 39), including infants (1, 5, 31), and is also present in other neonatal marsupial species, in particular the Virginia opossum (7) and Julia Creek dunnart (12). However, although the reflex could not be elicited in the tammar at just 6 h after birth, its presence in the early stages of the neonatal period is not surprising because the mechanisms underlying it appear to be developed well before birth (18). The strength of the HB reflex (quantified as IREXP) increases with age in the joey, which probably represents maturational changes in either central and/or peripheral vagal mechanisms (7, 37). Indeed, the functional maturation of the vagus nerve that accompanies myelinization provides an important peripheral influence during development in the opossum, which is thought to occur at ∼40 days postpartum (19). An equally feasible explanation is that the structural immaturity of the newborn marsupial lung might mean that, for a given inflation pressure, the firing response from the airway stretch receptors is attenuated compared with the more mature animals (9).
The postinspiratory pause is a prominent feature of the spontaneous breathing pattern in the newborns of a number of species including marsupials. The interpretation of the postinspiratory pause for characterization of breathing activities is valid in newborns as it has been clearly demonstrated that the pause results from interruption of the expiratory airflow, with the lung maintained above FRC and without diaphragmatic electromyographic activity (see Ref. 27 and references within). Observations of the breathing pattern of the newborn opossum indicated that the held inspirations could represent an early reflex control of breathing (7). This pattern of breathing is interesting because the longer Tp and, hence, sustained stretch receptor stimulation might be expected to facilitate desensitization and habituation of vagal inputs resulting in an earlier offset of the HB reflex (35). This suggestion is consistent with the age-related changes in the strength of the HB reflex in which younger joeys with a weaker reflex also exhibited the longest Tp (unpublished data).
Tb, ventilation, and metabolic rate. Hypothermia in the joey resulted in proportionate decreases in ventilation and metabolic rate. A similar finding has previously been reported in mildly anesthetized nonshivering adult rats and ground squirrels when Tb was reduced to 27°C (32), whereas in the unanesthetized state during severe hypothermia (Tb was lowered to 10 or 5°C) metabolic rate fell more than ventilation (44) and the ground squirrels experienced a relative hyperventilation (i.e., an increase in the convective requirement). This could suggest a limitation to oxygen extraction during severe hypothermia.
Lowering Tb in the joey resulted in a decrease in breathing rate as a result of an increase in Tp, whereas both T′e and Ti remained unaltered apart from 20°C when the breathing pattern became irregular and Ti and Tp markedly increased. Similarly, hypothermia in the rat and ground squirrel was associated with increases in expiratory time and the appearance of pauses in the breathing pattern (32). It is becoming increasingly apparent that breathing pattern in mammals is shaped by changes in metabolic rate, neural state, and temperature. Temperature can have profound effects. On cooling, for example, a Tb will eventually be reached when a hypothermia-induced respiratory arrest occurs. In the in vitro saggital slice preparation of the neonatal rat, cooling leads to an exponential slowing of the respiratory rhythm and ultimate arrest appears to occur at the level of the central rhythm-generating network (24). It is plausible that in the joey the instability of breathing as Tb approaches 20°C represents the failure of central mechanisms of respiratory control progressing toward some critical temperature or the lower thermal limits of the rhythm-generating network.
The HB reflex. In intact animals and infants, prolongation of Ti after occlusion is inversely related to the volume of the lung above FRC, whereas prolongation of Te is directly related to the volume of the lung above FRC (17). It is also well established that sustained inflation of the lung has little or no residual effect on Ti, whereas considerable prolongation of Te persists (42). In contrast, inflation of the lungs in infants, using a negative extrathoracic pressure (-6 cmH2O), resulted in a significant increase in Te, Ti, and Vt (15). These results support the suggestion that application of an inflation pressure results in an increase in FRC that resets tonic vagal activity and shifts the Vt-Ti relationship upward (36). Furthermore, because Te was prolonged with negative pressure, whereas Ti was relatively unchanged (36), it is apparent that, in contrast to earlier concepts of respiratory control (41), tonic stretch receptor stimulation must act to prolong Te independently of the effects on Ti. Finally, it has been shown (45) that Ti is prolonged in response to prolongation of the preceding Te, using stimuli applied only during expiration. This finding supports the notion that respiratory centers integrate information from the preceding breath. Given the above arguments and the small change in Ti observed with inflation at all temperatures and the possible reliance of Ti on the preceding Te, the HB reflex could not therefore be reliably determined by changes in Ti. Furthermore, discussion regarding the HB reflex is thus confined to our results for IREXP.
Tb and the HB reflex. Metabolic rate provides an important endogenous drive to breathe and is, therefore, an important determinant of the HB reflex (43). An increase in metabolic rate would be expected to result in an earlier offset of the ventilatory inhibition due to the rapidly ensuing hypoxic and hypercapnic drives, whereas a decrease in metabolic rate should prolong the HB reflex. Indeed, in the 2-day-old rat, after cold exposure, despite a fall in Tb, the increase in metabolic rate that accompanied thermogenesis reduced the strength of the HB reflex (25, 26, 43). In contrast, when Ta, and hence Tb, was lowered in the ectothermic joey, metabolic rate decreased and, opposite to the above suggestion, the strength of the HB reflex decreased. Such a finding supports the idea that Tb has a profound effect on the intensity of the HB reflex, at least in the absence of any thermogenic response. An obvious difference between the marsupial and eutherian newborn is that the eutherian newborn is capable of thermogenesis, albeit somewhat limited in the case of the rat (28). Nevertheless, the thermogenic effort of the newborn rat attenuates the fall in Tb that otherwise would have occurred and it seems reasonable to speculate that the effect of Tb on the HB reflex in the normoxic newborn rat was masked by both the increase in metabolic rate and the impaired drop in Tb that occurred on cold exposure.
Changes in temperature are known to affect the HB reflex through vagal reflexes (4, 38, 40). Normally, an increase in Vt is associated with a vagally mediated response from the pulmonary stretch receptors that shortens Ti. At a Tb of 20°C, despite an increase in Vt, ventilatory drive (Vt/Ti) in the joey was decreased due to a lengthening of Ti. A shortening in Ti has been previously reported with an increase in Tb (4). A similar result to that observed in the joey has been obtained in the vagotomized cat where loss of vagal afferents is associated with a deeper and slower breathing pattern (2). Together, these findings suggest that in the joey at 20°C vagal feedback on lung volume has almost been completely abolished. Interestingly, despite the apparent loss of feedback from the pulmonary stretch receptors and change in breathing pattern, pulmonary ventilation remains linked to metabolic rate, such that V̇e/V̇o2 is maintained constant. In fact, the sustained hyperpneic contribution to the hypoxia-induced hyperventilation during cold exposure implies that there was significant preservation of peripheral chemosensitivity. It might be reasonable to conclude, therefore, that the stretch receptors are relatively more temperature sensitive than the chemoreceptors. Consequently, at progressively lower Tb the inputs from the peripheral chemoreceptors exert a relatively greater influence on breathing compared with opposing inhibitory inputs. However, data from other ectotherms have revealed contradictory findings. In the alligator (6) and garter snake (14), for example, activity of the pulmonary stretch receptors decreased with temperature (Q10 ∼ 2.1). At the same time, the activity of CO2-sensitive receptors also decreased (Q10 ∼ 3.2). The difference in the Q10 values between these two receptors means that at lower Tbs the CO2-sensitive receptors played proportionately less of a role in ventilatory control; hence the pulmonary stretch receptors became relatively more important.
Tb, hypoxia, and the HB reflex. Hypoxia induced a hyperventilation (increase in V̇e/V̇o2) in the joey at all Tbs. In addition, the degree of hyperventilation and hyperpnea does not decline during hypothermia, suggesting, therefore, that the respiratory gain to hypoxic stimulus is not depressed and the sensitivity of the carotid chemoreceptor to hypoxia in the joey does not decrease at reduced Tb. Similar conclusions have been reached in the hypothermic rat (13, 21).
Hypoxia reduced the strength of the HB reflex in the joey at temperatures where it occurred, presumably because of the increased hypoxic drive. Such a marked drop in reflex strength might be unpredicted because in very young newborns vagal ventilatory inhibition is not particularly sensitive to the chemical drive to breathe (22) and, indeed, chemosensitivity is characteristically low in newborn mammals (25-27). It might also be expected that the hypoxic drive would be opposed by the decreased endogenous drive that is associated with the hypoxia-induced hypometabolism (29), which in turn would be expected to prolong the HB reflex. In the newborn rat, the HB reflex in hypoxia was essentially constant and independent of Tb (26).
The earlier suggestion that the increase in metabolic rate that occurred on cold exposure in normoxia had obscured the effect of Tb on the HB reflex in newborn rats would thus appear not to hold during hypoxia, because, despite changes in Tb accompanying changes in Ta, both metabolic rate and the HB reflex remained essentially constant (26). The lack of change in the HB reflex with hypoxia has been previously reported in 2-day-old rats (22) and could only occur if the drop in metabolic rate exactly offset the chemical stimuli from the peripheral chemoreceptors that should act to reduce the strength of the HB reflex. Perhaps the difference between the newborn rat and joey lies in the fact that the former maintains some degree of endothermy during hypoxia. The low Q10 (∼1.2 to 1.4) for V̇o2 reported in the newborn rat during hypoxia implies that the reduction in metabolic rate is not Q10 driven as it is for an ectotherm, where a Q10 of two or three would be expected. Instead, the metabolic rate observed in the newborn rat exposed to hypoxia reflects the attenuation of thermogenesis that accompanies hypoxia and changes in Tb are secondary, somewhat minor, and subsequently impart little influence on the HB reflex. On the other hand, in the ectothermic joey, Tb decides metabolic rate and appears paramount in determining the strength of the HB reflex, whereas exposure to hypoxia in the joey, while inducing a hypometabolism, has no effect on Tb, but does provide a strong chemical stimulus that acts to reduce the strength of the HB reflex.
In conclusion, vagal ventilatory mechanisms in the neonate may be affected by changes in Tb, especially in those newborns in which thermogenesis is deficient or absent. The present data in the ectothermic joey indicate that the decline in the HB reflex that occurs in moderate hypothermia is more than likely the result of a change in Tb having a direct effect on vagal mechanisms controlling breathing rather than a Q10-driven change in metabolic rate. In newborns in which thermogenesis is present, the increase in metabolism during cold exposure masks the effects that Tb per se have on the HB reflex. Overall, neither metabolic rate nor Tb should be ignored in studies that examine vagally mediated responses in the newborn mammal.
This work is supported by an Australian Research Council grant to P. B. Frappell. P. M. MacFarlane was the recipient of an Australian Postgraduate Research Award.
L. Masini is thanked for assistance in all aspects of this project. Appreciation is also extended to T. Clark for continued assistance in capturing animals.
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