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Controversy surrounds the respiratory responses to baroreceptor activation. Although many reflexes that effect respiration (e.g. chemoreflexes and nociceptive reflexes) frequently affect cardiovascular parameters, the effect of baroreflex stimulation within normal physiological limits is generally considered to affect only blood pressure and heart rate. Even though previous authors have reported that baroreceptor activation can affect respiratory activity, the effects on respiratory frequency and amplitude are highly variable, and changes in perfusion evoked by blood pressure manipulation could account for the observed effects. Here, we determined the respiratory effects of activating arterial baroreceptors by intravenous injection of phenylephrine or angiotensin II, or by electrical stimulation of the aortic depressor nerve (ADN). In urethane-anaesthetized vagotomized rats, 1, 2 and 4 second trains of tetanic ADN stimulation evoked 3.1 ± 1.1%, 11.2 ± 13.6% and 21.9 ± 8.9% increases in inspiratory (TI) time and 26.5 ± 18%, 23.4 ± 15.7% and 34.6 ± 20.9% increases in expiratory (TE) time respectively (P < 0.05 in both cases), but no effect on the amplitude of bursts recorded in the phrenic nerve. Similar effects were observed following pressor trials evoked by intravenous PE (TE: +26.1 ± 9.1%, P<0.01), but not Ang II. Intermittent ADN stimulation (single pulse, 1 Hz) significantly increased the variability of TI during periods of low respiratory drive (P < 0.05) without significantly affecting any other parameters. We propose that a specific baroreceptor-respiratory response exists that is independent of changes in blood flow. In contrast to the effects of baroreceptor stimulation on sympathetic nerve activity, the baro-respiratory response is subtle and highly dependent on respiratory drive.
The cardio-respiratory system is coordinated for the optimal delivery of oxygen and removal of carbon dioxide. Activation of many afferent pathways can affect both systems. This is perhaps most evident with the chemoreflex in which hypoxemia elicits increases in respiration and sympathetic motor discharge. This reflex, rarely active at rest, acts to shunt oxygen delivery from the skin and mesentery to maintain oxygen delivery to the central nervous system and other vital organs.
In contrast to the chemoreflex, activation of the baroreflex has highly variable effects on respiration that previous studies suggest is evoked primarily at high levels of baroreceptor activation. For example, Sapru and colleagues (1981) reported that in decerebrate non-vagotomized Wistar rats, aortic depressor nerve (ADN) stimulation completely arrests respiratory rhythm. In contrast, Miserocchi & Quinn (1980) found no effects of hemorrhagic hypotension on respiratory frequency in the cat, whereas Dove & Katona (1985) found that brief baroreceptor activation prolonged inspiratory (TI) and expiratory (TE) duration. Finally, Hayashi et al. (1993) found no effect of aortic nerve stimulation on respiratory frequency in urethane-anaesthetised Sprague Dawley rats. The differences observed seem to reflect, amongst other things, the species, strain, preparation and anesthesia.
There are two obvious ways in which baroreceptor input may influence respiratory output: on a beat- by-beat basis, pulsatile baroreceptor input may partially entrain respiratory rhythm (Tzeng et al. 2003; Tzeng et al. 2007a; Tzeng et al. 2007b) and modulate respiratory neuronal activity (Dick et al. 2004; Dick et al. 2005). Secondly, in response to systemic changes in blood pressure, barorespiratory interactions could decrease or increase respiratory activity during periods of hyper- or hypo-tension respectively, synchronizing ventilation with bloodflow within the lungs to optimize gas exchange (Bishop 1968; Bishop 1974; Fregosi 1994).
In this study we aimed to determine the effect of tetanic and intermittent ADN stimulation on phrenic nerve discharge amplitude and burst variability. We compared the effects of ADN stimulation to physiologic baroreceptor activation evoked by intravenous injection of the vasoconstrictors phenylephrine (PE) and angiotensin (Ang II). To control for potential central effects of PE and Ang II on the neural apparatus that underlies central respiratory drive, experiments were repeated in the same animals following acute bilateral sinoaortic denervation.
All experiments were approved by the Macquarie University Animal Care and Ethics Committee and were performed in accordance of the Australian guidelines for the care and use of animals. At the end of experiments animals were killed humanely by an intravenous bolus of 3M KCl (0.5 ml).
Experiments were conducted in 7 male Sprague Dawley rats (300–450 g) that were anaesthetized with 10% urethane (1.3 g kg−1 i.p.) and intubated. The right external jugular vein and right common carotid artery were cannulated in order to infuse drugs and record arterial blood pressure (AP) respectively. Core temperature was measured with a rectal probe and maintained at 37°C with a homeothermic blanket and an infra-red lamp. The anesthetic level was assessed at regular intervals and maintained at a depth that blocked increases in blood pressure and respiratory rate evoked by noxious pinch. When necessary, supplemental doses of anesthesia were given (10% urethane; 0.2–0.5 ml i.v.).
The left aortic depressor, phrenic, and splanchnic sympathetic nerves were isolated; the phrenic and splanchnic nerves were prepared for recording as previously described (Miyawaki et al. 1995). The aortic depressor nerve (ADN) was identified by recording its characteristic pulse-modulated activity. The ADN was separated from surrounding tissue and cut as distally as possible (10–15 mm caudal to the carotid bifurcation) and the proximal end tied with a short length of 10/0 suture. The vagi were transected bilaterally; the right ADN was cut with the right vagus.
Following surgery, rats were positioned prone in a stereotaxic frame with a clamp on lumbar vertebral processes to elevate the animal. A 5 % dextrose solution was infused continuously (5 ml kg−1 hr−1, i.v.) to ensure hydration during the experiment. Neuromuscular blockade was evoked by pancuronium bromide (0.2 mg hr−1 i.v.). Rats were artificially ventilated and end-tidal CO2 was maintained at 3.5 – 4.5 % (measured using a Capstar-100 (CWE Inc, USA sampling at 30 ml/min). Arterial blood gases were measured once preparation was complete and once or twice during the experiment to ensure appropriate ventilation. Acceptable blood pH was 7.2–7.45. The electrocardiogram (ECG) was recorded from leads placed on forelimbs and referred to the reference electrode. Wound margins and exposed tissue (except nerves) were protected by agar, and a reference electrode was inserted into nuchal muscle.
Two polyethylene cannulae were fixed using cyanoacrylate glue with their tips externally placed at the left and right carotid bifurcations. Acute barodenervation was achieved by bilateral injection of bupivacaine (5 – 10 mg), a long-lasting local anesthetic (Farnham et al. 2009). Barodenervation was judged successful if the pulse modulation of splanchnic sympathetic nerve activity (sSNA) was blocked after bupivacaine infusion.
Nerves were mounted on bipolar silver electrodes and embedded in silicon rubber (Elastosil®, Wacker Chemie AG). Signals from the phrenic and splanchnic nerves were amplified, filtered (0.1 – 2 kHz bandpass), and sampled (5 kHz) with a CED Micro1401 Mark II and Spike 2 version 6 software. Data were rectified, smoothed (5 ms time constant), and normalized between maximal (sSNA: immediately after i.v. KCl; phrenic nerve activity (PNA): maximal response to 10 s hypoxia) and post-mortem levels. Phrenic nerve frequency (PNf), TI and TE were derived offline. The duration and timing of stimuli were controlled by a programmable Spike 2 script (Lidierth 2005); stimulus intensity was determined using an isolated stimulator.
We determined the effect of single and tetanic ADN stimulation on sSNA, AP, PNA and PNf. ADN stimulus intensity was set at three times the threshold at which 1 strains (3 ms pulse duration, 50 Hz pulse frequency) evoked a decreases in sSNA and/or AP. Threshold was typically between 2.5 and 5V..
First, we assessed the effects of 1, 2 and 4 strains of tetanic stimulation on PNf, TI and TE. We then examined the effects of intermittent ADN stimulation (1 Hz, 300 stimuli) on sSNA and PNA amplitude and TI and TE at baseline conditions (median end-tidal CO2, (EtCO2): 4.8%; arterial blood pH 7.3 ± 0.02), near apnoeic threshold (EtCO2: 3.9%), and during respiratory acidosis (EtCO2: 5.5%). Steady state respiratory pattern was recorded for at least 5 min prior to testing the responses to ADN stimulation.
Finally, single ADN stimuli were triggered by the rising envelope of PNA with or without 0.4 or 0.8 s delays in order to examine the effects of ADN stimulation at different phases of the respiratory cycle.
Responses to bolus intravenous injections of phenylephrine (PE, 245 nmol) and angiotensin II (Ang II, 35 pmol) in 0.3 ml saline were assessed. These doses were chosen on the basis that they elicit equipressor responses (McMullan et al. 2007). The maximal effects of acute pressor ramps evoked by PE or Ang II injection were assessed on PNf, TI and TE. Once blood pressure returned to baseline, a 10-min period was allowed for recovery. To test whether any effects observed were specifically due to activation of baroreceptors, trials were repeated following acute barodenervation.
The effects of 1, 2 and 4 strains of tetanic ADN stimulation on TI and TE duration were compared to the relevant values that immediately preceded stimulation. For stimuli that spanned more than one phase of TI or TE, baseline values were compared to the final phase, even if stimulation terminated before the end of that phase. Data were normalized with respect to baseline values and the effect of tetanic ADN stimulation on TI or TE was considered significant if the results of 1-way ANOVA returned P < 0.05.
To assess the effects of single ADN stimuli triggered by inspiration, and at 0.4 or 0.8 s following inspiration, the phase of TI or TE that occurred directly after stimulation was measured. Mean and coefficient of variation (CV) of TI and TE were compared to data recorded over the 5-min period prior to stimulation using 1-way ANOVA. If the stimulus occurred midway through a phase of TI or TE, that phase was included.
To assess respiratory pattern variability, we constructed Poincaré plots of TI and TE for 200 breaths prior to and during intermittent ADN stimulation. The region of the sample that contained 95% of data points was calculated for each plot, as described by Sokal and Rohlf (2001), and the area of the 95% region and the mean was measured and compared between groups. 2-way ANOVA was used to compare the effect of ADN stimulation at different levels of central respiratory drive. Student’s t-test with Bonferroni’s correction post-test was used to assess differences when indicated by a statistically significant 2-way ANOVA result (P<0.05).
Peak effects of PE and Ang II on PNf, TI, TE and AP were compared to the average of the 30-s period immediately prior to agent injection and expressed relative to baseline. The distribution of each set was tested using the Kolmogorov-Smirnov test; changes from baseline that came from normally distributed sets were tested using a 1-sample t-test; data that came from a non-Gaussian distribution were assessed using the Wilcoxon signed rank test. (The only set that did not fit a normal distribution was PE-TE). The PE vs. Ang II dataset was expanded by inclusion of data previously obtained using the same preparation (McMullan et al. 2007). Five experiments from the previous dataset were selected at random; the effects of PE and Ang II on respiratory variables were reanalyzed as described above. None of the parameters from the previously recorded dataset were significantly different from the current dataset, so both were combined to increase the statistical power of the observations.
Sympathetic nerve activity was measured in 6 of 7 animals. All 3 modes of ADN activation (intermittent, tetanic and inspiratory-triggered) evoked decreases in sSNA; obvious effects on AP were only seen following tetanic stimulation (Figure 1). One second of ADN stimulation at 50 Hz reduced AP from 113±4 mmHg to 88±6 mmHg (P< 0.01). The response to intermittent stimulation was transient and had a short latency; waveform averages indicated inhibition began at approximately 100 ms after the onset of the stimulus, had two distinct nadirs, one at approximately 150 ms and the other at 275 ms (Figure 2), and resolved after approximately 600 ms. The inhibitory effect on sSNA was present no matter when the stimulus was delivered during the respiratory cycle (Figure 3). Both intravenous PE and Ang II evoked large increases in AP but different responses in sSNA, which became quiescent after PE but remained active after Ang II (Figure 5), as previously reported (McMullan et al. 2007).
In comparison with the effects on sSNA and AP, the effect of ADN stimulation on PNA appeared modest. Trains of ADN stimulation did not reduce the amplitude of PNA (Figure 1A), regardless of their duration or stimulus intensity (data not shown). Similarly, stimulus-triggered averages revealed no effect of intermittent ADN stimulation on PNA amplitude, whether the stimulus occurred irrespective of respiratory phase (Figure 2) or at a specific delay after the onset of inspiration (Figure 3).
Both tetanic and intermittent ADN stimulation had effects on respiratory timing. Tetanic ADN stimulation caused a significant increase in TI and TE that immediately recovered following cessation of stimulation and was proportional to stimulus duration (Figure 1B). TI increased by 3.1 ± 1.1%, 11.2 ± 13.6% and 21.9 ± 8.9% by 1, 2 and 4 s 50 Hz trains respectively (P < 0.05). Tetanic ADN stimulation caused a greater prolongation of TE compared to TI (P < 0.05) that was less obviously dependent on stimulus duration; 1, 2 and 4 strains caused TE to increase by 26.5 ± 18%, 23.4 ± 15.7% and 34.6 ± 20.9% respectively (P < 0.05). We noticed that the degree to which tetanic ADN stimulation increased TI depended to a certain extent on the phase of the respiratory cycle at which stimulation commenced –stimulation that coincided with the start of inspiration tended to have a greater effect on TI than trials in which stimulation was applied during TE (data not shown). This sensitivity was not observed in the effect of ADN stimulation on TE.
As tetanic ADN stimulation lengthened the respiratory period without affecting amplitude of PNA, we hypothesized that timing parameters may be affected preferentially. Poincaré plots showed that intermittent ADN stimulation significantly increased the variability of TI (P < 0.05, Figure 4) but no other parameters. The effect was only significant at low central respiratory drive (i.e. during high ventilation). ADN stimulation did not significantly alter mean PNf, TI or TE. As expected, PNf increased with respiratory drive (P < 0.01); due to reductions in TI (P < 0.001), and TE (P = 0.074).
Intravenous PE or Ang II caused a large increase in mean AP from pre-injection values of 112 ± 8 and 116 ± 7 mmHg to peaks of 198 ± 9 and 201 ± 12 mmHg respectively (n = 10). Pressor responses evoked by PE consistently caused a decrease in PNf (−22.4 ± 3.8%, P<0.01), mediated by an increase in TE (+26.1 ± 9.1%, P<0.01). There was no consistent effect of PE-evoked hypertension on TI. In contrast, pressor ramps evoked by Ang II had much more variable effects; PNf, TI and TE were frequently perturbed during Ang II trials (see Fig. 5), but the effects were as likely to be tachypneic as bradypneic.
In two experiments with successful acute barodenervations, the effects of PE-evoked pressor trials on PNf and TE were reversed (Figure 5).
The main findings of this study are that activation of high-pressure baroreceptors by either intermittent or tetanic ADN stimulation or by pressor trials evoked by PE (but not Ang II) evokes distinct effects on respiratory timing but not on phrenic nerve amplitude. The way in which these effects manifested varied, but were generally consistent with bradypneic effects. For example, tetanic ADN stimulation and intravenous PE, both of which evoked sSNA quiescence, reduced PNf by prolongation of TE. In contrast, intermittent ADN stimulation, which transiently inhibited rather than silenced sSNA, increased the variability of TI during low respiratory drive but did not exert statistically significant effects on any of the other parameters measured.
The results observed here are likely due to selective activation of barosensory afferents. Care was taken to minimize the risk of stimulus currents inadvertently activating other nearby afferent nerves such as those contained in the recurrent laryngeal or vagus nerves, which are known to strongly activate cardiorespiratory reflexes. Long portions (10 – 15 mm caudal to the carotid bifurcation) of ADN were surgically exposed to reduce proximity to other nerves, and the entire nerve and stimulating electrode assembly were embedded in silicon rubber, which has a very high electrical resistivity (1016 Ω-cm).
Although the ADN has traditionally been seen as a ‘purely barosensory nerve’ in the rat (Sapru et al. 1977; Kobayashi et al. 1999), there is strong evidence that a minority of ADN afferents are chemosensitive in function (Brophy et al. 1999). However, even if chemoreceptors were activated by ADN stimulation, chemoreceptor reflexes are known to increase phrenic nerve frequency and amplitude and sympathoexcitation, whereas the opposite effects were observed in the present study.
One potential source of error in conducting studies of this kind is that a specific stimulus (baroreceptor stimulation) could evoke changes in respiratory behavior by a secondary mechanism. For example, raised AP evoked by intravenous vasoconstrictors would increase perfusion of the carotid body and brainstem, potentially reducing chemoreceptor input, and therefore reducing respiratory drive. We have avoided these effects by using a range of stimuli that have contrasting secondary effects. Intravenous PE or Ang II result in large pressor effects; tetanic ADN stimulation reduces AP; and intermittent ADN stimulation did not affect AP. Significantly; all three approaches evoked qualitatively similar effects on respiratory activity.
One surprising finding of the current study is that the bradypneic effects evoked by PE pressor trials were not mimicked by Ang II, despite PE and Ang II raising AP by the same degree. A possible explanation could be the different hemodynamic effects evoked by PE and Ang II, which may result in the activation of different combinations of vascular mechanoreceptors (other than arterial baroreceptors). In addition to its effects on resistance vessels, PE increases venous tone, raising venous, pulmonary artery, and circulatory filling pressure, at least in anaesthetized dogs (Appleton et al. 1985). Similar effects of PE, but not Ang II, have been observed in conscious humans (Shenker et al. 1988). Thus pressor trials evoked by PE, but not Ang II, would be expected to activate pulmonary and atrial stretch receptors, suggesting a mechanism by which the two vasonconstrictors could generate different afferent signals. However, most pulmonary and atrial mechanoreceptor afferents travel in the vagus nerve (Cowley 1992; Moore et al. 2004), which was cut in our preparation. Furthermore, pulmonary baroreceptors evoke sympathoexcitatory and tachypneic effects (McMahon et al. 2000). Considering the bradypneic effects evoked by PE trials were reversed in experiments in which acute barodenervation was successful, it seems likely that the respiratory effects evoked PE were specifically evoked by arterial baroreceptor activation. We and others have previously shown that equivalent pressor trials evoked by PE and Ang II activate baroreceptors in the ADN to the same degree (Lumbers et al. 1979; Guo et al. 1984; McMullan et al. 2007), so this is not the source of the difference. It therefore seems that a difference in the central effects evoked by PE compared to Ang II may underlie these effects. There is strong evidence that circulating Ang II dampens central baroreflex pathways (Guo et al. 1984; Matsukawa et al. 1991; McMullan et al. 2007); it could simply be that, in addition to attenuating barosensory input to presympathetic pathways, Ang II also attenuates the barosensory input that drives the barorespiratory response.
In the current study, 1, 2 or 4 strains of ADN stimulation at 50 Hz evoked complete baroinhibition of sSNA and relatively subtle bradypneic effects. Both TI and TE were prolonged by tetanic stimulation by up to 30% of their baseline values; we found that the effects on TI were much more sensitive to train duration than TE. In contrast, we found no effects on PNA amplitude. Our findings are qualitatively similar, but vastly different in magnitude, to those reported by Sapru et al. (1981), who found that comparable intensities (although longer trains) of tetanic ADN stimulation were capable of abolishing PNA and both inspiratory and excitatory volleys in the recurrent laryngeal nerve in decerebrate rats. Although Sapru et al. report that phrenic quiescence was not always evoked when stimulus frequency was dropped below 50 Hz; it is unlikely that the differences observed in the current study are due to differences in stimulus intensity. Our results are almost an order of magnitude more subtle than those observed by Sapru et al., who saw 50–60% reductions in PNf in response to even the mildest ADN stimulation (e.g. 2 Hz stimulation). The stimuli used in the current study reduced sSNA to levels comparable to those recorded post-mortem, suggesting that 50 Hz stimulation maximally activated the baroreceptor reflex. This being the case, it seems unlikely that any difference in the stimulation protocols used in the two studies underlies the differences in the results. The use of decerebrate (Sapru et al. 1981) or anesthetized preparations (current study) may underlie these differences.
It seems that the most methodologically relevant study to ours was conducted by Hayashi et al. (1993), who used the same strain of rat, the same anaesthetic agent, and comparable parameters for stimulation of the ADN. In contrast to our findings, Hayashi et al. were unable to demonstrate any effect of ADN stimulation on respiratory frequency.
We found similar effects on respiratory rhythm when large pressor responses were evoked by bolus injection of PE, which may be considered a more physiologic approach than electrical stimulation of the ADN. Other investigators have activated carotid sinus baroreceptors by inflation of a blind sac, which increased TI modestly and TE markedly in the dog (Hopp et al. 1998). The converse was also evoked by reducing sac pressure. In their study, the authors showed convincing examples of both effects and noted that tidal volume was not significantly affected. Similar findings in the dog have been reported by other groups (Brunner et al. 1982; Dove et al. 1985). In contrast, using the same approach, Maass-Moreno and Katona lengthened TE but shortened TI in the cat (Maass-Moreno et al. 1989). In a recent study in piglets by Curran and Leiter (2007), reductions in PNA amplitude and frequency during pressor trials evoked by aortic balloon inflation were accentuated by intravenous injection of a 5-HT1A agonist. The authors tested their hypothesis that this was due to inhibition of sympathetic premotor neurons in the RVLM and caudal raphé nucleus by dialyzing 8-hydroxydipropylaminotetralin into the medulla; and subsequently showed this hypothesis to be unlikely.
In the current study, intermittent ADN stimulation evoked subtle but consistent effects on the variability of TE. Interestingly, this effect was only unmasked when respiratory drive was reduced by establishing a relative alkalosis by hyperventilation. To our knowledge, no previous study has investigated the effects that altering respiratory drive may have on respiratory reflexes evoked by baroreceptor stimulation. The sensitivity of barorespiratory responses to changes in respiratory drive contrasts to the resilience of sympathetic baroreflexes, which persist regardless of excitatory respiratory (Figures 2C +D) or nociceptive inputs (Li et al. 1998). We speculate that this is due to the increased synchrony of firing that occurs in respiratory networks in response to reductions in pH. Extracellular pH and chemoreceptor input are the most important determinants of central respiratory drive at eupnea; thus removal of central and chemoreceptor drives by oxygen-enriched hyperventilation would increase the influence that other inputs exert on the network. In contrast, a strong respiratory drive may dominant barosensory input to the respiratory system and mask barorespiratory responses. Such effects may underlie the variability of respiratory responses (particularly pertaining to TI) reported in the literature. Baroreceptor activation has been reported to have bradypneic (Brunner et al. 1982; Dove et al. 1985) or no effects (Grunstein et al. 1975; Miserocchi et al. 1980) on respiratory frequency, and to either lengthen (Brunner et al. 1982; Dove et al. 1985) or shorten TI (Maass-Moreno et al. 1989).
In the anaesthetized vagotomized rat, we find that baroreceptor stimulation can evoke relatively subtle decreases in respiratory frequency that manifest as an increase of the expiratory period or its variability, with variable effects on inspiratory period and no effects on amplitude. Taken together, these findings suggest that baroreceptors provide a weak input to the circuits that generate respiratory rhythm, but not inspiratory amplitude. At eupnoea, the influence exerted by baroreceptor inputs is minimal, but when baroreceptor input is very high, as might happen during extreme exertion or during periods of very low ventilatory drive, such as deep sleep, it appears that baroreceptor input may modulate respiratory rhythmogenesis. It remains to be determined if this response plays a significant role in homeostasis in such situations, although physiological roles of similar mechanisms such as atrial stretch have previously been described (Chenuel et al. 2006).
Work in the Authors’ laboratories is supported by grants from Macquarie University and by the National Health and Medical Research Council of Australia (211023, 211196), the Garnett Passe and Rodney Williams Memorial Foundation, and by the National Institutes of Health of the United States (NHLBI HL080318)
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