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Effects of baroreceptor activation on respiratory variability in rat
Respiratory Physiology & Neurobiology 166 (2009) 80–86
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Effects of baroreceptor activation on respiratory variability in rat Simon McMullan a,1 , Thomas E. Dick b,1 , Melissa M.J. Farnham a , Paul M. Pilowsky a,∗ a
Australian School of Advanced Medicine, Macquarie University, 3 Innovation Road, Sydney, NSW 2109, Australia Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Case Western Reserve University, 44106 Cleveland, OH, United States b
a r t i c l e
i n f o
Article history: Accepted 2 February 2009 Keywords: Respiratory drive Sympathetic nerve activity Aortic nerve Angiotensin
a b s t r a c t 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-anesthetized vagotomized rats, 1, 2 and 4 s 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. © 2009 Elsevier B.V. All rights reserved.
1. Introduction 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 et al. (1981) reported that in decerebrate non-vagotomized Wistar rats, aortic depressor nerve (ADN) stimulation completely arrests respiratory rhythm. In contrast, Miserocchi and Quinn (1980) found no effects of
∗ Corresponding author. Tel.: +61 2 9850 4015; fax: +61 2 9850 4010. E-mail address: [email protected] (P.M. Pilowsky). 1 These authors contributed equally to this study. 1569-9048/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2009.02.006
hemorrhagic hypotension on respiratory frequency in the cat, whereas Dove and 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-anesthetized 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, 2007a,b) and modulate respiratory neuronal activity (Dick and Morris, 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 hypotension, respectively, synchronizing ventilation with bloodflow within the lungs to optimize gas exchange (Bishop, 1968, 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
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Fig. 1. Effect of tetanic aortic depressor nerve (ADN) stimulation on respiratory frequency. (A) Experimental trace shows the effect of tetanic ADN stimulation on phrenic nerve frequency (PNf), splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity (PNA) and arterial blood pressure (AP). 50 Hz ADN stimulation causes a profound inhibition of sympathetic tone, resulting in the decrease in AP, and a reduction in PNf but no change in the amplitude of PNA. Note: stimulus artifacts have been removed from sSNA and PNA traces to enhance clarity. (B) Pooled data from seven rats show that tetanic ADN stimulation causes a significant increase of inspiratory (TI) and expiratory (TE) time that increases with the duration of stimulation. *P < 0.05.
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. 2. Materials and methods 2.1. Animal preparation 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 3 M KCl (0.5 ml). Experiments were conducted in seven male Sprague–Dawley rats (300–450 g) that were anesthetized 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 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 h−1 , i.v.) to ensure hydration during the experiment. Neuromuscular blockade was evoked by pancuronium bromide (0.2 mg h−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 pro-
Fig. 2. (A) Pooled (n = 6) stimulus-triggered averages of sSNA and PNA responses to intermittent ADN stimulation (*). ADN stimulation inhibited sSNA but not PNA. Solid black data = mean; dashed grey lines = SEM. (B) Experimental trace shows the effects of intermittent ADN stimulation (*) on sSNA, PNA, ECG and AP. Although the sympathoinhibitory effect of ADN stimulation is evident from the raw data, no other effects are apparent. ADN stimuli are at 1 s intervals.
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2.3. Nerve recording and stimulation 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. 2.4. Experimental protocol 2.4.1. Effect of ADN stimulation on sympathetic and phrenic nerve activities 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 s trains (3 ms pulse duration, 50 Hz pulse frequency) evoked a decreases in sSNA and/or AP. Threshold was typically between 2.5 and 5 V. First, we assessed the effects of 1, 2 and 4 s trains 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 endtidal 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.
Fig. 3. Effect of intermittent ADN stimulation triggered at different phases of the respiratory cycle on respiratory output. (A)–(D) show pooled inspiratory-triggered waveform averages from six experiments. (A) Steady-state sSNA shows considerable respiratory modulation. (B) ADN stimulation triggered by the rising envelope of PNA causes a powerful inhibition of sSNA without any effect on PNA. Similar effects are seen when stimulation is delivered 0.4 s (C) or 0.8 s (D) after inspiration. (E) Intermittent ADN stimulation triggered at or 0.4 s or 0.8 s after inspiration had no effect on the duration of TI or TE, or on the coefficient of variation (CV) of TI or TE.
tected by agar, and a reference electrode was inserted into nuchal muscle. 2.2. Acute barodenervation 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 and Pilowsky, 2009). Barodenervation was judged successful if the pulse modulation of splanchnic sympathetic nerve activity (sSNA) was blocked after bupivacaine infusion.
2.4.2. Effect of pharmacologic increases in blood pressure on sympathetic and phrenic nerve activities Responses to bolus intravenous injections of phenylephrine (245 nmol) and angiotensin 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 10min period was allowed for recovery. To test whether any effects observed were specifically due to activation of baroreceptors, trials were repeated following acute barodenervation. 2.5. Data analysis 2.5.1. Effects of tetanic ADN stimulation The effects of 1, 2 and 4 s trains 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 one-way ANOVA returned P < 0.05. 2.5.2. Effects of inspiratory-triggered ADN stimulation 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
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Fig. 4. Poincaré plots of TI (A) and TE (B) at baseline (grey circles) and during intermittent ADN stimulation (black circles) at three different levels of respiratory drive induced by varying ventilation (high: left column; mid: center column; low: right column) recorded in a single experiment. For each Poincaré plot the coordinate of the mean value is indicated by a cross-hair. The 95% boundary of each series is designated by a dashed grey ellipse. (C) Pooled data (n = 6) show that mean baseline PNf and TI (open circles; left panels) are closely related to central respiratory drive and are not affected by ADN stimulation (closed circles). However, the variability of TI is greatly increased by ADN stimulation (middle right panel). This effect is only unmasked by reducing respiratory drive. Similar trends are seen in TE, although the differences are not statistically significant. ***P < 0.001, **P < 0.01 vs. respiratory drive, † P < 0.05 baseline vs. ADN stimulation.
recorded over the 5-min period prior to stimulation using one-way ANOVA. If the stimulus occurred midway through a phase of TI or TE, that phase was included. 2.5.3. Poincaré plots 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. Two-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 two-way ANOVA result (P < 0.05). 2.5.4. Effects of pressor trials evoked by PE and Ang II on respiratory variables 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 dis-
tribution were assessed using the Wilcoxon signed rank test. (The only set that did not fit a normal distribution was PE–TE.) The PE versus 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. 3. Results 3.1. Effect of ADN stimulation on splanchnic sympathetic nerve activity and arterial blood pressure Sympathetic nerve activity was measured in six of seven animals. All three modes of ADN activation (intermittent, tetanic and inspiratory-triggered) evoked decreases in sSNA; obvious effects on AP were only seen following tetanic stimulation (Fig. 1). One second of ADN stimulation at 50 Hz reduced AP from 113 ± 4 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
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Fig. 5. Equipressor AP trials evoked by intravenous phenylephrine (PE) and angiotensin II (Ang II) have different effects on respiratory drive. (A) Data from a single experiment shows the effects of large, acute pressor responses on PNf, TI, TE, sSNA, PNA and AP. In the example shown, both agents evoke large increases in AP that correspond with reductions in TI and increased TE and baroinhibition of sSNA (note that the effect of Ang II on sSNA is less profound than that of PE). PE reduces PNf, whereas Ang II has no effect (top trace). (B) Pooled data from 10 experiments shows the effects of PE- and Ang II-evoked pressor trials on PNf, TI and TE. PE trials (open circles) significantly decrease PNf and increase TE compared to baseline, but have no effect on TI. Although equal in magnitude, pressor ramps evoked by Ang II did not reproducibly affect any respiratory parameters (closed circles), although, as demonstrated in (A), changes in TI and TE were frequently seen. In two experiments the effects of PE-evoked pressor trials on PNf and TE were reversed by acute barodenervation (B; grey circles). Horizontal lines indicate mean values, except for PE–TE, in which median is indicated. Barodenervation was evoked by application of bupivacaine at the carotid bifurcation via previously implanted catheters and was judged complete if baseline pulse modulation of sSNA was disrupted (C). **P < 0.01.
and the other at 275 ms (Fig. 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 (Fig. 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 (Fig. 5), as previously reported (McMullan et al., 2007). 3.2. Effect of baroreceptor activation on phrenic nerve activity 3.2.1. Effects on phrenic nerve discharge amplitude 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 (Fig. 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 (Fig. 2) or at a specific delay after the onset of inspiration (Fig. 3). 3.2.2. Effects on phrenic nerve discharge timing 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 (Fig. 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 s trains 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 inspi-
ration 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, Fig. 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). 3.2.3. Effects of PE and Ang II on phrenic nerve discharge 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 (Fig. 5). 4. Discussion The main findings of this study are that activation of highpressure 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
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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.
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 and Abboud, 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.
4.1. Technical considerations
4.2. Comparisons with other work
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 -cm). Although the ADN has traditionally been seen as a ‘purely barosensory nerve’ in the rat (Sapru and Krieger, 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 anesthetized 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 and Abboud, 1984; McMullan et al., 2007), so this is not the source of the difference. It therefore
In the current study, 1, 2 or 4 s trains 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 anesthetic 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 and Seagard, 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 and Katona, 1985). In contrast, using the same approach, Maass-Moreno and Katona lengthened TE but shortened TI in the cat (MaassMoreno and Katona, 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
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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 (Fig. 2C and 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 and Katona, 1985) or no effects (Grunstein et al., 1975; Miserocchi and Quinn, 1980) on respiratory frequency, and to either lengthen (Brunner et al., 1982; Dove and Katona, 1985) or shorten TI (Maass-Moreno and Katona, 1989). 4.3. Conclusion In the anesthetized 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). Acknowledgements 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). References Appleton, C., Olajos, M., Morkin, E., Goldman, S., 1985. Alpha-1 adrenergic control of the venous circulation in intact dogs. J. Pharmacol. Exp. Ther. 233, 729–734. Bishop, B., 1968. Diaphragm and abdominal muscle activity during induced hypotension. J. Appl. Physiol. 25, 73–79. Bishop, B., 1974. Carotid baroreceptor modulation of diaphragm and abdominal muscle activity in the cat. J. Appl. Physiol. 36, 12–19. Brophy, S., Ford, T.W., Carey, M., Jones, J.F., 1999. Activity of aortic chemoreceptors in the anaesthetized rat. J. Physiol. 514, 821–828. Brunner, M.J., Sussman, M.S., Greene, A.S., Kallman, C.H., Shoukas, A.A., 1982. Carotid sinus baroreceptor reflex control of respiration. Circ. Res. 51, 624–636.
Chenuel, B.J., Smith, C.A., Skatrud, J.B., Henderson, K.S., Dempsey, J.A., 2006. Increased propensity for apnea in response to acute elevations in left atrial pressure during sleep in the dog. J. Appl. Physiol. 101, 76–83. Cowley Jr., A.W., 1992. Long-term control of arterial blood pressure. Physiol. Rev. 72, 231–300. Curran, A.K., Leiter, J.C., 2007. Baroreceptor-mediated inhibition of respiration after peripheral and central administration of a 5-HT1A receptor agonist in neonatal piglets. Exp. Physiol. 92, 757–767. Dick, T.E., Morris, K.F., 2004. Quantitative analysis of cardiovascular modulation in respiratory neural activity. J. Physiol. 556, 959–970. Dick, T.E., Shannon, R., Lindsey, B.G., Nuding, S.C., Segers, L.S., Baekey, D.M., Morris, K.F., 2005. Arterial pulse modulated activity is expressed in respiratory neural output. J. Appl. Physiol. 99, 691–698. Dove, E.L., Katona, P.G., 1985. Respiratory effects of brief baroreceptor stimuli in the anesthetized dog. J. Appl. Physiol. 59, 1258–1265. Farnham, M.M.J., Pilowsky, P.M., 2009. Local anaesthetics for acute reversible blockade of the sympathetic baroreceptor reflex in the rat. J. Neurosci. Methods 179, 58–62. Fregosi, R.F., 1994. Changes in the neural drive to abdominal expiratory muscles in hemorrhagic hypotension. Am. J. Physiol. 266, H2423–H2429. Grunstein, M.M., Derenne, J.P., Milic-Emili, J., 1975. Control of depth and frequency of breathing during baroreceptor stimulation in cats. J. Appl. Physiol. 39, 395–404. Guo, G.B., Abboud, F.M., 1984. Angiotensin II attenuates baroreflex control of heart rate and sympathetic activity. Am. J. Physiol. 246, H80–H89. Hayashi, F., Coles, S.K., Bach, K.B., Mitchell, G.S., McCrimmon, D.R., 1993. Timedependent phrenic nerve responses to carotid afferent activation: intact vs. decerebellate rats. Am. J. Physiol. 265, R811–R819. Hopp, F.A., Seagard, J.L., 1998. Respiratory responses to selective blockade of carotid sinus baroreceptors in the dog. Am. J. Physiol. 275, R10–R18. Kobayashi, M., Cheng, Z.B., Tanaka, K., Nosaka, S., 1999. Is the aortic depressor nerve involved in arterial chemoreflexes in rats? J. Auton. Nerv. Syst. 78, 38–48. Li, W.M., Liu, X., Kumada, M., Sato, A., 1998. Excitation of baroreceptors depresses A- and C-components of the somato-cardiac sympathetic reflex in anesthetized rats. Jpn. J. Physiol. 48, 261–266. Lidierth, M., 2005. Pulser: user-friendly, graphical user-interface based software for controlling stimuli during data acquisition with Spike2 for Windows. J. Neurosci. Methods 141, 243–250. Lumbers, E.R., McCloskey, D.I., Potter, E.K., 1979. Inhibition by angiotensin II of baroreceptor-evoked activity in cardiac vagal efferent nerves in the dog. J. Physiol. 294, 69–80. Maass-Moreno, R., Katona, P.G., 1989. Species dependence of baroreceptor effects on ventilation in the cat and the dog. J. Appl. Physiol. 67, 2116–2124. Matsukawa, T., Gotoh, E., Minamisawa, K., Kihara, M., Ueda, S., Shionoiri, H., Ishii, M., 1991. Effects of intravenous infusions of angiotensin II on muscle sympathetic nerve activity in humans. Am. J. Physiol. 261, R690–R696. McMahon, N.C., Drinkhill, M.J., Myers, D.S., Hainsworth, R., 2000. Reflex responses from the main pulmonary artery and bifurcation in anaesthetised dogs. Exp. Physiol. 85, 411–420. McMullan, S., Goodchild, A.K., Pilowsky, P.M., 2007. Circulating angiotensin II attenuates the sympathetic baroreflex by reducing the barosensitivity of medullary cardiovascular neurones. J. Physiol. (Lond.) 582, 711–722. Miserocchi, G., Quinn, B., 1980. Control of breathing during acute hemorrhage in anesthetized cats. Respir. Physiol. 41, 289–305. Miyawaki, T., Pilowsky, P., Sun, Q.J., Minson, J., Suzuki, S., Arnolda, L., Llewellyn-Smith, I., Chalmers, J., 1995. Central inspiration increases barosensitivity of neurons in rat rostral ventrolateral medulla. Am. J. Physiol. 268, R909–R918. Moore, J.P., Hainsworth, R., Drinkhill, M.J., 2004. Pulmonary arterial distension and vagal afferent nerve activity in anaesthetized dogs. J. Physiol. 555, 805–814. Sapru, H.N., Gonzalez, E., Krieger, A.J., 1981. Aortic nerve stimulation in the rat: cardiovascular and respiratory responses. Brain Res. Bull. 6, 393–398. Sapru, H.N., Krieger, A.J., 1977. Carotid and aortic chemoreceptor function in the rat. J. Appl. Physiol. 42, 344–348. Shenker, Y., Bates, E.R., Egan, B.H., Hammoud, J., Grekin, R.J., 1988. Effect of vasopressors on atrial natriuretic factor and hemodynamic function in humans. Hypertension 12, 20–25. Sokal, R., Rohlf, F., 2001. Correlation. In: Biometry: The Principles and Practice of Statistics in Biological Research. W.H. Freeman and Company, New York, pp. 586–593. Tzeng, Y.C., Larsen, P.D., Galletly, D.C., 2003. Cardioventilatory coupling in resting human subjects. Exp. Physiol. 88, 775–782. Tzeng, Y.C., Larsen, P.D., Galletly, D.C., 2007a. Effects of hypercapnia and hypoxemia on respiratory sinus arrhythmia in conscious humans during spontaneous respiration. Am. J. Physiol. Heart Circ. Physiol. 292, H2397–H2407. Tzeng, Y.C., Larsen, P.D., Galletly, D.C., 2007b. Mechanism of cardioventilatory coupling: insights from cardiac pacing, vagotomy, and sinoaortic denervation in the anesthetized rat. Am. J. Physiol. Heart Circ. Physiol. 292, H1967–H1977.
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Effects of baroreceptor activation on respiratory variability in rat Simon McMullan a,1 , Thomas E. Dick b,1 , Melissa M.J. Farnham a , Paul M. Pilowsky a,∗ a
Australian School of Advanced Medicine, Macquarie University, 3 Innovation Road, Sydney, NSW 2109, Australia Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Case Western Reserve University, 44106 Cleveland, OH, United States b
a r t i c l e
i n f o
Article history: Accepted 2 February 2009 Keywords: Respiratory drive Sympathetic nerve activity Aortic nerve Angiotensin
a b s t r a c t 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-anesthetized vagotomized rats, 1, 2 and 4 s 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. © 2009 Elsevier B.V. All rights reserved.
1. Introduction 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 et al. (1981) reported that in decerebrate non-vagotomized Wistar rats, aortic depressor nerve (ADN) stimulation completely arrests respiratory rhythm. In contrast, Miserocchi and Quinn (1980) found no effects of
∗ Corresponding author. Tel.: +61 2 9850 4015; fax: +61 2 9850 4010. E-mail address: [email protected] (P.M. Pilowsky). 1 These authors contributed equally to this study. 1569-9048/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2009.02.006
hemorrhagic hypotension on respiratory frequency in the cat, whereas Dove and 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-anesthetized 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, 2007a,b) and modulate respiratory neuronal activity (Dick and Morris, 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 hypotension, respectively, synchronizing ventilation with bloodflow within the lungs to optimize gas exchange (Bishop, 1968, 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
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Fig. 1. Effect of tetanic aortic depressor nerve (ADN) stimulation on respiratory frequency. (A) Experimental trace shows the effect of tetanic ADN stimulation on phrenic nerve frequency (PNf), splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity (PNA) and arterial blood pressure (AP). 50 Hz ADN stimulation causes a profound inhibition of sympathetic tone, resulting in the decrease in AP, and a reduction in PNf but no change in the amplitude of PNA. Note: stimulus artifacts have been removed from sSNA and PNA traces to enhance clarity. (B) Pooled data from seven rats show that tetanic ADN stimulation causes a significant increase of inspiratory (TI) and expiratory (TE) time that increases with the duration of stimulation. *P < 0.05.
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. 2. Materials and methods 2.1. Animal preparation 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 3 M KCl (0.5 ml). Experiments were conducted in seven male Sprague–Dawley rats (300–450 g) that were anesthetized 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 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 h−1 , i.v.) to ensure hydration during the experiment. Neuromuscular blockade was evoked by pancuronium bromide (0.2 mg h−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 pro-
Fig. 2. (A) Pooled (n = 6) stimulus-triggered averages of sSNA and PNA responses to intermittent ADN stimulation (*). ADN stimulation inhibited sSNA but not PNA. Solid black data = mean; dashed grey lines = SEM. (B) Experimental trace shows the effects of intermittent ADN stimulation (*) on sSNA, PNA, ECG and AP. Although the sympathoinhibitory effect of ADN stimulation is evident from the raw data, no other effects are apparent. ADN stimuli are at 1 s intervals.
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2.3. Nerve recording and stimulation 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. 2.4. Experimental protocol 2.4.1. Effect of ADN stimulation on sympathetic and phrenic nerve activities 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 s trains (3 ms pulse duration, 50 Hz pulse frequency) evoked a decreases in sSNA and/or AP. Threshold was typically between 2.5 and 5 V. First, we assessed the effects of 1, 2 and 4 s trains 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 endtidal 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.
Fig. 3. Effect of intermittent ADN stimulation triggered at different phases of the respiratory cycle on respiratory output. (A)–(D) show pooled inspiratory-triggered waveform averages from six experiments. (A) Steady-state sSNA shows considerable respiratory modulation. (B) ADN stimulation triggered by the rising envelope of PNA causes a powerful inhibition of sSNA without any effect on PNA. Similar effects are seen when stimulation is delivered 0.4 s (C) or 0.8 s (D) after inspiration. (E) Intermittent ADN stimulation triggered at or 0.4 s or 0.8 s after inspiration had no effect on the duration of TI or TE, or on the coefficient of variation (CV) of TI or TE.
tected by agar, and a reference electrode was inserted into nuchal muscle. 2.2. Acute barodenervation 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 and Pilowsky, 2009). Barodenervation was judged successful if the pulse modulation of splanchnic sympathetic nerve activity (sSNA) was blocked after bupivacaine infusion.
2.4.2. Effect of pharmacologic increases in blood pressure on sympathetic and phrenic nerve activities Responses to bolus intravenous injections of phenylephrine (245 nmol) and angiotensin 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 10min period was allowed for recovery. To test whether any effects observed were specifically due to activation of baroreceptors, trials were repeated following acute barodenervation. 2.5. Data analysis 2.5.1. Effects of tetanic ADN stimulation The effects of 1, 2 and 4 s trains 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 one-way ANOVA returned P < 0.05. 2.5.2. Effects of inspiratory-triggered ADN stimulation 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
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Fig. 4. Poincaré plots of TI (A) and TE (B) at baseline (grey circles) and during intermittent ADN stimulation (black circles) at three different levels of respiratory drive induced by varying ventilation (high: left column; mid: center column; low: right column) recorded in a single experiment. For each Poincaré plot the coordinate of the mean value is indicated by a cross-hair. The 95% boundary of each series is designated by a dashed grey ellipse. (C) Pooled data (n = 6) show that mean baseline PNf and TI (open circles; left panels) are closely related to central respiratory drive and are not affected by ADN stimulation (closed circles). However, the variability of TI is greatly increased by ADN stimulation (middle right panel). This effect is only unmasked by reducing respiratory drive. Similar trends are seen in TE, although the differences are not statistically significant. ***P < 0.001, **P < 0.01 vs. respiratory drive, † P < 0.05 baseline vs. ADN stimulation.
recorded over the 5-min period prior to stimulation using one-way ANOVA. If the stimulus occurred midway through a phase of TI or TE, that phase was included. 2.5.3. Poincaré plots 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. Two-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 two-way ANOVA result (P < 0.05). 2.5.4. Effects of pressor trials evoked by PE and Ang II on respiratory variables 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 dis-
tribution were assessed using the Wilcoxon signed rank test. (The only set that did not fit a normal distribution was PE–TE.) The PE versus 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. 3. Results 3.1. Effect of ADN stimulation on splanchnic sympathetic nerve activity and arterial blood pressure Sympathetic nerve activity was measured in six of seven animals. All three modes of ADN activation (intermittent, tetanic and inspiratory-triggered) evoked decreases in sSNA; obvious effects on AP were only seen following tetanic stimulation (Fig. 1). One second of ADN stimulation at 50 Hz reduced AP from 113 ± 4 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
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Fig. 5. Equipressor AP trials evoked by intravenous phenylephrine (PE) and angiotensin II (Ang II) have different effects on respiratory drive. (A) Data from a single experiment shows the effects of large, acute pressor responses on PNf, TI, TE, sSNA, PNA and AP. In the example shown, both agents evoke large increases in AP that correspond with reductions in TI and increased TE and baroinhibition of sSNA (note that the effect of Ang II on sSNA is less profound than that of PE). PE reduces PNf, whereas Ang II has no effect (top trace). (B) Pooled data from 10 experiments shows the effects of PE- and Ang II-evoked pressor trials on PNf, TI and TE. PE trials (open circles) significantly decrease PNf and increase TE compared to baseline, but have no effect on TI. Although equal in magnitude, pressor ramps evoked by Ang II did not reproducibly affect any respiratory parameters (closed circles), although, as demonstrated in (A), changes in TI and TE were frequently seen. In two experiments the effects of PE-evoked pressor trials on PNf and TE were reversed by acute barodenervation (B; grey circles). Horizontal lines indicate mean values, except for PE–TE, in which median is indicated. Barodenervation was evoked by application of bupivacaine at the carotid bifurcation via previously implanted catheters and was judged complete if baseline pulse modulation of sSNA was disrupted (C). **P < 0.01.
and the other at 275 ms (Fig. 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 (Fig. 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 (Fig. 5), as previously reported (McMullan et al., 2007). 3.2. Effect of baroreceptor activation on phrenic nerve activity 3.2.1. Effects on phrenic nerve discharge amplitude 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 (Fig. 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 (Fig. 2) or at a specific delay after the onset of inspiration (Fig. 3). 3.2.2. Effects on phrenic nerve discharge timing 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 (Fig. 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 s trains 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 inspi-
ration 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, Fig. 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). 3.2.3. Effects of PE and Ang II on phrenic nerve discharge 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 (Fig. 5). 4. Discussion The main findings of this study are that activation of highpressure 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
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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.
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 and Abboud, 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.
4.1. Technical considerations
4.2. Comparisons with other work
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 -cm). Although the ADN has traditionally been seen as a ‘purely barosensory nerve’ in the rat (Sapru and Krieger, 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 anesthetized 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 and Abboud, 1984; McMullan et al., 2007), so this is not the source of the difference. It therefore
In the current study, 1, 2 or 4 s trains 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 anesthetic 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 and Seagard, 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 and Katona, 1985). In contrast, using the same approach, Maass-Moreno and Katona lengthened TE but shortened TI in the cat (MaassMoreno and Katona, 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
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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 (Fig. 2C and 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 and Katona, 1985) or no effects (Grunstein et al., 1975; Miserocchi and Quinn, 1980) on respiratory frequency, and to either lengthen (Brunner et al., 1982; Dove and Katona, 1985) or shorten TI (Maass-Moreno and Katona, 1989). 4.3. Conclusion In the anesthetized 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). Acknowledgements 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). References Appleton, C., Olajos, M., Morkin, E., Goldman, S., 1985. Alpha-1 adrenergic control of the venous circulation in intact dogs. J. Pharmacol. Exp. Ther. 233, 729–734. Bishop, B., 1968. Diaphragm and abdominal muscle activity during induced hypotension. J. Appl. Physiol. 25, 73–79. Bishop, B., 1974. Carotid baroreceptor modulation of diaphragm and abdominal muscle activity in the cat. J. Appl. Physiol. 36, 12–19. Brophy, S., Ford, T.W., Carey, M., Jones, J.F., 1999. Activity of aortic chemoreceptors in the anaesthetized rat. J. Physiol. 514, 821–828. Brunner, M.J., Sussman, M.S., Greene, A.S., Kallman, C.H., Shoukas, A.A., 1982. Carotid sinus baroreceptor reflex control of respiration. Circ. Res. 51, 624–636.
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