Cardiovascular responses to nasal water flow in rats are unaffected by chemoreceptor drive P. F. McCULLOCH AND N. H. WEST Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0 WO, Canada McCulloch, P. F., and N. H. West. Cardiovascular responses to nasal water flow in rats are unaffected by chemoreceptor drive. Am. J. Physiol. 263 (Regulatory Integrative Cornp. Physiol. 32): Rl049-R1056, 1992.-Peripheral chemoreceptors generally play a limited role in the initial development of diving bradycardia in mammals. However, T. F. Huang and Y. I. Peng (Jpn. J. Physiol. 26: 395-401, 1976) reported that peripheral chemoreceptors are very important for manifestation of the diving response in conscious rats. The objectives of this study were to reinvestigate those findings and determine whether the cardiovascular responses to simulated diving in the rat were potentiated during preexisting hypoxia or hypercapnia. Responses to simulated diving were elicited by nasal water flow with concurrent apnea in paralyzed, artificially ventilated Sprague-Dawley rats anesthetized with Innovar. The experiments show that nasal stimulation in the rat results in rapid bradycardia and hypotension and that these responses are not due to laryngeal stimulation. The data also suggest that chemoreceptors do not play a role in the initiation of the responses to simulated diving in rats and that preexisting chemoreceptor drive does not alter the cardiovascular responses. Additionally, we found that concomitant expiratory apnea is necessary to sustain the profound initial cardiovascular changes induced by nasal water flow. trigeminal nerve stimulation; laryngeal nerve stimulation; mammalian diving response; diving bradycardia HUANG AND PENG (6) reported that peripheral chemoreceptors are important for the manifestation of bradycardia during involuntary submersion in the conscious rat. Carotid sinus chemoreceptor denervation or intrasinusal injection of acetic acid eliminated the cardiovascular response to simulated diving. Daly (2) reviewed mammalian breath-hold diving mechanisms and concluded that chemoreceptors exert their influence primarily in the latter stages of prolonged breath-hold diving. For example, in seals and rabbits the carotid body chemoreceptors do not contribute to the bradycardia until the increasing asphyxial stages of the apneic period (3, 10). In muskrats chemoreceptors contribute to the diving bradycardia only after 20 s of submergence (4). Thus Huang and Peng’s conclusion that chemoreceptors could have an important role in the initial development of the diving response in the rat seems unlikely. Huang and Peng (6) also found that sectioning of the recurrent or superior laryngeal nerves did not affect the cardiovascular response to submersion in the rat. They used this finding to support their conclusion that the chemoreceptors play an important role in the diving response. The objectives of this study were to reinvestigate the findings of Huang and Peng (6) concerning the importance of chemoreceptor stimulation in the diving response in the rat and to determine whether the cardiovascular responses to simulated diving are potentiated when there is a preexisting state of chemoreceptor stim0363-6119/92
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ulation, as during hypoxia or hypercapnia. A third objective was to reevaluate the importance of laryngeal receptor stimulation in generating the response. The cardiovascular responses to diving were simulated by nasal water flow with concurrent apnea in anesthetized, paralyzed, and artificially ventilated rats. Additionally, the cardiovascular effects of apnea were separated from those of nasal water flow in normoxic rats. METHODS Experiments were performed on male Sprague-Dawley rats (370-589 g). The procedures were approved by the Animal Care Committee of the University of Saskatchewan and conform to the standards of the Canadian Council on Animal Care. Surgical
Preparation
Rats were anesthetized with Innovar (Innovar-Vet, MTC Pharmaceuticals, Cambridge, Ontario; 0.15-0.2 ml/kg im, diluted to a 10% solution), after initial inhalation induction with methoxyflurane (Metofane, MTC Pharmaceuticals). Half-dose injections of Innovar were given hourly to maintain anesthesia. A femoral artery was cannulated (PE-50, Clay Adams, Parsippany, NJ) for arterial blood pressure measurement and arterial blood sampling. The ipsilateral femoral vein was cannulated for injection of a paralytic agent. Copper leads were inserted under the skin to record electrocardiogram (ECG), and a cardiotachometer (type 9857B, Beckman Instruments, Schiller Park, IL) was used to monitor heart rate. Body temperature was maintained at 38 k 1°C with a heating pad (Harvard Animal Temperature Control Unit, Ealing Scientific, St. Laurent, Quebec). The rats were placed in a supine position, the trachea was exposed through a ventral incision, and oral- and caudal-facing tracheal cannulas (PE-205, 1.57 mm ID) were inserted. The rats were then paralyzed with d-tubocurarine (Sigma, St. Louis, MO; 2 mg/kg iv), and ventilated through the caudal-facing cannula [model CTP-930, CWE, Ardmore, PA; respiratory fretidal volume (V,) adjusted for body wt; quency = 70 min-l, range 2.6-3.8 ml]. The oral-facing cannula was positioned with its tip just caudal to the epiglottis. The previously retracted submaxillary glands were repositioned, and the skin incision was tightly closed with wound clips (Autoclip, Clay Adams). In those trials involving water stimulation of the nasal region, room temperature tap water was pumped through the oral-facing cannula at 10 ml/min (model 50lU/R, Watson-Marlowe, Falmouth, England). If the water exited through the mouth and not through the external nares, the submaxillary glands and oral-facing cannula were repositioned, the incision was reclosed, and the trial was repeated. Experimental
Procedures
Nasal water flow trials with concurrent expiratory apnea (initiated by turning off the ventilator) were performed initially with the recurrent laryngeal nerves (RLN) and superior laryngeal nerves (SLN) intact. The water flow through the oralfacing cannula could not directly stimulate the receptors with afferents in the RLN and SLN because the cannula tip extended rostra1 to the larynx. However, stimulation of the laryngeal
0 1992 the American
Physiological
Society
R1049
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R1050
NASAL
STIMULATION
nerves was possible as water flow through the cannula could produce mechanical disturbances to stimulate the larynx. To eliminate possible laryngeal receptor stimulation, the RLN and SLN were sectioned bilaterally, and the nasal water flow with apnea trials were repeated. To investigate possible interactions between the cardiovascular effects of stimulation of the receptors in the nasal region and chemoreceptor drive, rats were ventilated to produce four groups with differing partial pressures of arterial 0, (Pao ) and CO, (Pace,). Arterial blood gases for the four groups were: >roup 1, Pao = ~80 mmHg and Pace = ~35 mmHg (normoxic and normocapnic condition); group 2”, Pao > 100 mmHg and Pace < 30 mmHg (hyperoxic and hypocapnic condition, designed to reduce peripheral and central chemoreceptor drive); group 3, Pao > 100 mmHg and Pace > 50 mmHg (hyperoxic and hypercapnic condition, designed to stimulate the chemoreceptors through an increased Pace,); and group 4, Pao, < 40 mmHg and Pace < 30 mmHg (hypoxic and hypocapnic condition, designed to stimulate the peripheral chemoreceptors through a lowered Pao,). Inspired gases were mixed with a gas mixing pump (Digamix, model M/300 A, Wosthoff, Bochum, Germany). Arterial pH, PO,, and PCO~ were monitored every 15-30 min (IL Micro 213 pH/blood gas analyzer, Instrumentation Laboratory, Lexington, MA), and V, and inspired fractions of N2, 02, and CO, were adjusted to ensure that blood gas values were appropriate for each group (Table 1). To separate the cardiac effects of apnea alone from those of nasal water flow during nasal water flow plus concurrent apnea, three experimental stimuli were used: apnea was expiratory apnea (initiated by turning off the ventilator), nasal water flow was nasal water flow through the oral-facing cannula without concurrent apnea, and nasal water flow plus concurrent apnea consisted of simultaneous nasal water flow and expiratory apnea. Six rats were used in each group, with each rat receiving three or more trials of each stimuli. All trials lasted 10 s. Five minutes separated each trial, and any water in the oral-facing cannula and nasal passages was cleared by blowing air through the cannula at the end of each trial. ECGs and arterial pressures were recorded on a Beckman R511A chart recorder.
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Means for each animal were calculated by determining average heart rate (HR) and mean arterial blood pressure (MABP) from all trials. Instantaneous HR was calculated from cardiac intervals, and MABP was calculated from pulsatile blood pressure traces (diastolic plus one-third pulse pressure). Grand means for each experimental stimulus were calculated by averaging the means from all animals. Values reported in text and in Figs. 2 and 4-8 are grand means * SE, with HR in beats per minute and MABP in millimeters Hg. Control HR and MABP were calculated by averaging three pretrial scores (at -5, -2.5, and 0 s) immediately preceding the onset of the experimental stimuli for each animal. Control HR and MABP between groups 1, 3, and 4 were found to be significantly different from each other (Table 2). To facilitate comparisons between these groups,
I
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Fig. 1. Original chart recordings of nasal water flow plus concomitant expiratory apnea before (A) and after (B) the recurrent (RLN) and superior laryngeal nerves (SLN) were transected. Top to bottom: electrocardiogram (ECG), heart rate (HR), pulsatile arterial blood pressure (BP), ventilation (up, inspiration; down, expiration), event marker (down, onset of nasal water flow).
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2.5
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10.0
(s)
Fig. 2. HR (A) and mean arterial blood pressure (MABP) (B) responses (&SE) to nasal water flow plus expiratory apnea (n = 6) before and after RLN and SLN were cut. At no time were responses significantly different from each other. BPM, beats per minute.
Table 1. Arterial pH, Po2, and Pco2 values for four groups ventilated with different gases Group
1
2 3 4
PK
7.580t0.010" 7.646kO.021" 7.355k0.020h 7.551t0.011"
Pa0,,
mmHg
76.822.8" 106.3t2.6d l17.6k6Jd 35.9t1.2c
Pwo,,
mm&
34.lk1.3" 28.4kO.7' 55.8k2.6" 25.2tl.0f
Values are means t SE; n = 6 for each group. Groups: I, normoxic-normocapnic; 2, hyperoxic-hypocapnic; 3, hyperoxic-hypercapnic; hypoxic-hypocapnic. Value significantly different from: a arterial pH (pH,) values of groups 2 and 3; b all other pH, values; c all other arterial (Pao,) values; d Pao, values of groups 1 and 4; e all other arterial PCO~ (Paoo,) values; or f Pace, values of groups 1 and 3.
4, PO,
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NASAL
Table 2. Control
heart rate and mean arterial
STIMULATION
blood pressure
Apnea
Nasal
Rl051
IN RATS
for groups Water
1, 3, and 4 Nasal
Flow
Water
Flow
Plus
Apnea
Group HR
1 3 4
468.2t3.2* 393.2t2.7 364.8t2.1
MABP
88.7t0.3 88.3t0.3 46.8+0.1-f
HR
MABP
454.0t3.9” 384.7t5.9 370.3t7.4
91.9tO.l 92.1t0.3 46.4+0.5-f
Values are means t SE; n = 6 for each group. Groups: 1, normoxic-normocapnic; rate (HR) and mean arterial blood pressure (MABP) are beats/min and mmHg, and nasal water flow plus apnea, control HR and MABP were not significantly 2 groups, t MABP significantly lower than other 2 groups.
raw scores were transformed into a percent of control. This transformation did not affect the normality of the data distribution (13). Statistical analyses were performed with a computer package (SYSTAT, Systat, Evanston, IL). Student’s t test and one- and two-way analysis of variance (ANOVA) with repeated measures were employed, as required (7, 13). Statistical significance was reached if P was < 0.05. In the case of significant F values, Tukey’s honestly significant difference or Fisher’s least significant difference a posteriori tests were performed to determine significant differences among group means.
Nasal water flow plus concurrent apnea produced a rapid and substantial drop in HR and a rapid decrease in MABP (Fig. 1A). With the RLN and SLN intact, there was a 76.7% drop in HR (462.3 t 4.7 to 107.8 t 14.0 beats/min at 2.5 s) and a 41.1% decrease in MABP (89.7 t 0.3 to 52.8 t 4.4 mmHg) (Fig. 2). These responseswere not observed if the water exited through the oral cavity and not the external nares. The cardiovascular responses to nasal water flow plus apnea did not differ after the RLN and SLN were cut (Fig. 1B). HR decreased by 80.1% (451.0 t 5.8 to 89.9 t 7.5 beats/min at 2.5 s), and MABP decreased by 36.8% (89.9 t 0.3 to 56.8 t 4.9 mmHg at 2.5 s) (Fig. 2). This indicates that stimulation of afferents in the RLN and SLN is not required for elicitation of the cardiovascular responses to nasal water flow.
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3, hyperoxic-hypercapnic; 4, hypoxic-hypocapnic. Units of heart respectively. Across experimental stimuli of apnea, nasal water flow, different within each group. * HR significantly greater than other
In the normoxic-normocapnic condition, nasal water flow plus apnea was more effective in inducing cardiovascular changes than was either of the two stimuli applied individually (Fig. 3). Throughout the duration of the trials, HR from the three stimuli were statistically different (Fig. 4). HR decreased the most during nasal water flow plus apnea, from 457.0 t 0.8 to 101.9 t 15.3 beats/min at 2.5 s. In comparison, HR decreased from 454.0 t 3.9 to 250.0 t 47.8 b eat s/ min at 2.5 s during nasal water flow, but only from 468.2 t 3.2 to 446.6 t 4.3 during apnea. During both nasal water flow and nasal water flow plus apnea, HR increased after the initial fall, whereas during apnea there was a slight decrease in HR throughout the duration of the trials. MABP for all three conditions was statistically different within the first 5 s, but MABP during nasal water flow then increased to a level not significantly different from that during apnea (Fig. 4). Throughout the duration of the trials, nasal water flow plus apnea MABP remained lower than control MABP. Apnea MABP increased above control; nasal water flow MABP initially decreased but then rose above control.
Cardiovascular Effects of Nasal Water Flow Plus Apnea
A
Role of Chemoreceptor Drive During Nasal Stimulation Withdrawal of chemoreceptor drive. The results from group 2 (hyperoxic-hypocapnic) are similar to group 1 (normoxic-normocapnic) (Fig. 5). During nasal water flow plus apnea, HR decreased immediately from 475.7 t 6.3 to 93.8 t 11.9 beats/min at 2.5 s and remained at that
B
C
1
a Fig. 3. Original chart recordings of apnea (A), nasal water flow (B), and nasal water flow plus apnea (C) in a normoxic-normocapnic rat. Apnea resulted in minimal HR and BP changes. Nasal water flow alone resulted in immediate but transient bradycardia and hypotension. Nasal water flow plus apnea resulted in an immediate bradycardia and hypotension that was sustained for duration of stimulus. Top to bottom: ECG, HR, pulsatile arterial BP, ventilation (up, inspiration; down, expiration), event marker (down, onset of stimulus).
'c; 100
2% mg
50 0I
VENTILATION EVENT MARKER
MABP
91.4tl.O 87.7t0.2 47.3+0.3-F
Importance of Concurrent Apnea During Nasal Stimulation
RESULTS
ECG
HR
457.0t0.8* 389.3t4.2 364.5t9.5
n
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Rl052
NASAL
STIMULATION
IN RATS
A
700
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Apnea
0
Nasal
water
flow
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plus
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Fig. 4. HR (A) and MABP (B) responses (GE) to apnea, nasal water flow, and nasal water flow plus apnea for group 1 (normoxic-normocapnit) (n = 6). Nasal water flow plus apnea resulted in the greatest changes in HR and MABP, and apnea resulted in minimal changes. * Response significantly different from other 2 responses at that time; ** response significantly different from both apnea and nasal water flow responses at that time.
level for the duration of the stimulus. MABP decreased from 97.4 t 1.1 to 68.8 t 5.7 mmHg and remained at -75% of control MABP. With apnea, HR again showed little change while MABP increased above control levels. During nasal water flow, HR fell from 481.7 t 7.0 to 209.0 t 41.4 beats/min at 2.5 s but then increased to 358.4 t 39.6 beats/min at 7.5 s. MABP again initially decreased but then rose above control. HRs for the three experimental stimuli were statistically different from each other throughout the duration of the trials. Nasal water flow plus apnea MABP was significantly less than the other two stimuli. Chemoreceptor stimulation through preexisting hypercapnia or hypoxia. The cardiovascular responses to apnea, nasal water flow, and nasal water flow plus apnea for group 3 (hyperoxic-hypercapnic) and group 4 (hypoxichypocapnic) were compared with the responses for group 1 (normoxic-normocapnic). Control HR and MABP values for the three groups are presented in Table 2. Within each group, control HR and MABP were not significantly different across the three experimental stimuli. However, control HR for the three experimental stimuli was signif-
80
m
60
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40
k s
20 O
CONTROL
2.5
5.0
Time
7.5
10.0
(s)
Fig. 5. HR (A) and MABP (B) responses (GE) to apnea, nasal water flow, and nasal water flow plus apnea for group 2 (hyperoxic-hypocapnit) (n = 6). Nasal water flow plus apnea resulted in the greatest changes in HR and MABP, and apnea resulted in minimal changes. HR and BP responses to nasal water flow alone showed an initial decrease but then became attenuated throughout duration of stimulus. * Response significantly different from other 2 responses at that time; ** response significantly different from both apnea and nasal water flow responses at that time.
icantly higher for group 1 than for groups 3 and 4. Control MABP was significantly lower for group 4 than for the other two conditions. Because of these differences in control values, HR and MABP were standardized to facilitate comparisons between groups. The cardiovascular responses to apnea, nasal water flow, and nasal water flow plus apnea for the three groups are presented in Figs. 6-8. HR remained constant and MABP increased above 100% in groups 1 and 3 during apnea (Fig. 6). In group 4, HR fell steadily to reach 36% of control at 10 s. MABP initially increased, but by 10 s MABP had decreased to 84%. During nasal water flow alone HR fell to -55% in all three groups by 2.5 s (Fig. 7). HR then increased and remained at -80% for the duration of the water flow. MABP initially decreased, but by 10 s all were above 100%. No response from the three groups was significantly different from any other during nasal water flow. During nasal water flow plus apnea (Fig.
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NASAL
V
Group
1
Group
3
Group
4
STIMULATION
IN
RATS
R1053
Fig. 6. Percent (GE) of control HR (A) and MABP (B) during apnea in group 1 (normoxic-normocapnic), group 3 (hyperoxic-hypercapnic), and group 4 (hypoxic-hypocapnit) (n = 6 for each group). Values are percent of control presented in Table 2. Group 4 showed a progressive decrease in HR and a decrease in MABP toward end of apneic period. * Response significantly different from the other 2 groups at that time; ** response significantly different from group 1 at that time.
t
2 0
60
$
50
I
CONTROL
I
I
I
2.5
5.0
7.5
Time
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1
10.0
(s)
8), HR decreased to 20% at 2.5 s in all three groups and rose slightly but remained at a decreased level (~45%) for the remainder of the stimulus duration. MABP fell to -67% in all three groups but then rose to -80%. Group 4 MABP decreased by a lesser extent than the other two groups, but only at 5 s were the differences between groups significant. DISCUSSION
Huang and Peng (6) reported that bilateral section of the carotid sinus nerve or destruction of the carotid bodies by intrasinusal injection of acetic acid in the conscious rat eliminated the bradycardic response to simulated diving. They concluded that the peripheral chemoreceptors were important in the manifestation of the cardiovascular responses to diving in the rat. This conclusion is surprising, as others have found that the carotid body chemoreceptors appear to be of little significance in initiation of the bradycardic response in both aquatic (muskrat, mink, and seal) and terrestrial mammals (rabbit) (3-5, 10, 12). The main conclusion of this study is that peripheral chemoreceptor stimulation is unnecessary for the initiation of the cardiovascular responses to simulated diving in the rat. This contradicts the findings of Huang and
Peng (6) but is in accordance with other mammalian literature regarding the role of chemoreceptor drive in breath-hold diving (3-5, 12). The main evidence for this conclusion is that after the chemoreceptors were offloaded during hyperoxic-hypocapnic ventilation, the cardiovascular responses to apnea, nasal water flow, and nasal water flow plus apnea were similar to those observed during the normoxic-normocapnic condition. Increased Pao or decreased Pace reduces peripheral chemoreceptor activity (8), thus functionally denervating the peripheral chemoreceptors. This method is preferable to other methods of reducing chemoreceptor input because all chemoreceptive sites, including carotid and aortic bodies and central chemoreceptors, become simultaneously and reversibly off-loaded. Attenuated chemoreceptor drive thus does not alter the cardiovascular responses to nasal stimulation observed during normal chemoreceptor drive. Further support for our conclusion that chemoreceptors do not play a role in the initial dive response of the rat is provided by cardiovascular responses to nasal water flow plus apnea during hyperoxic-hypercapnic and hypoxic-hypocapnic ventilation. If chemoreceptor drive is important for the manifestation of the diving response in
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R1054
t
NASAL
STIMULATION
IN RATS
100 0
’
80
2 i 0
60
Fig. 7. Percent (GE) of control HR (A) and MABP (B) during nasal water flow in group 1 (normoxic-normocapnit), group 3 (hyperoxic-hypercapnic), and group 4 (hypoxic-hypocapnic) (n = 6 for each group). Values are percent of control presen ted in Table 2. Responses were not significantly different from each other at any time. ;
100 0
4
90
T
2 0 0 y0
80 70
CONTROL
2.5
5.0 Time (s)
7.5
the rat, then increased chemoreceptor drive resulting from preexisting hypoxia or hypercapnia (8) should potentiate the cardiovascular responses to nasal water flow plus apnea. Our results indicate that this is not the case. The cardiovascular responses to nasal water flow plus apnea during normal chemoreceptor drive were no different from the responses observed when there was an increased chemoreceptor drive due to preexisting hypoxia or hypercapnia. Therefore, chemoreceptor stimulation through preexisting hypoxia or hypercapnia does not alter the cardiovascular responses to nasal water flow plus apnea. Preexisting hypoxic chemoreceptor drive did, however, alter the cardiac response to expiratory apnea. During hypoxic hypocapnia, HR fell progressively to 36% of its control, whereas HR in the normoxic and hypercapnic conditions changed only slightly. Slowly developing bradycardia during lung deflation in normoxic mink is partially alleviated by hyperoxia, suggesting an influence from the peripheral chemoreceptors (12). Carotid body stimulation contributes to the bradycardia toward the end of long-duration simulated dives in seals, helping to maintain the bradycardia during the developing asphyxia (3). Together these results indicate that chemoreceptor stimulation, either through progressive or preexisting hy-
10.0
poxia, results in a slowly developing bradycardia. Upon closer examination, the results of Huang and Peng (6) appear to be more of a response to progressive hypoxia than to upper airway stimulation. In the present study the short duration of the stimulus presumably precluded the development of substantial progressive hypoxia; thus only during preexisting hypoxia did the hypoxic chemoreceptor drive result in a slowly developing bradycardia during expiratory apnea. A further finding is that the rapid and intense bradycardia that develops during nasal water flow with concomitant apnea in anesthetized, paralyzed, and artificially ventilated rats is not due to stimulation of the recurrent or superior laryngeal nerves. The bradycardic response was unaltered after sectioning of these nerves. We concluded that the response appears to be due to stimulation of the trigeminal nerves innervating the nasal mucosa because no cardiovascular response was elicited when the water exited through the oral cavity rather than the external nares. Placing the animal in a supine position facilitated the water flow through the nares, and it was very important to clear as much water as possible from the nasal passages between trials to prevent a diminution of the response with repeated trials. The cardiovascular responses to nasal water flow plus
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NASAL
-t:
STIMULATION
IN
R1055
RATS
80
0
$
60
Fig. 8. Percent (&SE) of control HR (A) and MABP (B) during nasal water flow plus apnea in group 1 (normoxicnormocapnic), group 3 (hyperoxic-hypercapnic), and group 4 (hypoxic-hypocapnic) (n = 6 for each group). Values are percent of control presented in Table 2. * Response was significantly different from group 3 at that time.
CONTROL
2.5
5.0
Time
7.5
10.0
(s)
apnea included both bradycardia and hypotension. After laryngeal nerves were cut (Fig. Z), HR decreased by 80.1%, but MABP decreased by only 36.8%. If one assumes no change in stroke volume, decreased HR would cause cardiac output to fall, which would lead to a decreased MABP. However, the MABP decrease was only one-half that of the HR decrease. This suggests that nasal water flow plus apnea results in both a bradycardia and a peripheral vasoconstriction. A point worth addressing is the validity of comparing the cardiovascular responses of an anesthe tized, paralyzed, and ventilated preparation with those of the conscious animal (6). It has been shown that input from the cerebral cortex in conscious muskrats influences the magnitude of diving bradycardia by ~20% (9). Furthermore, although anesthesia can affect central interactions between afferent receptor groups (1 I), Innovar has been shown to have only mod&ate effects on cardiovascular function and reflexes (1). At least two species of mammals, apart from the rat, exhibit a similar rapid-onset bradycardia during both conscious diving (voluntary or involuntary submersion ) and nasal water flow when anesthetized, paralyzed, and ventilated (4 12) 9suggesting that the same receptor-driven response is initiated in both cases.
In summary, chemoreceptor stimulation does not play a major role in the initiation of diving bradycardia in the rat. Reduction of chemoreceptor stimulation, or chemoreceptor stimulation through preexisting hypoxia or hypercapnia, did not alter the cardiovascular responses to trigeminal stimulation by nasal water flow. This refutes the conclusions of Huang and Peng (6) but is consistent with other work in mammals. We found concomitant expiratory apnea during nasal stimulation necessary to sustain the substantial cardiovascular changes, although nasal stimulation without concurrent apnea still causes a large initial bradycardia and hypotension. Nasal water flow stimulates trigeminal receptors in the upper respiratory tract, and the-physiological responses to this stimulus were not due to activation of receptors with afferent fibers in the superior or recurrent laryngeal nerves. We thank Dr. Mark Evered for critical comments on the manuscript. This work was supported by grants from the Medical Research Council of Canada to N. H. West, and by Heart and Stroke Foundation of Saskatchewan Traineeships and a University of Saskatchewan Graduate Scholarship to P. F. McCulloch. Address for reprint requests: P. McCulloch, Dept. of Physiology, College of Medicine, Univ. of Saskatchewan, Saskatoon, Saskatchewan S7N OWO, Canada. Received
21 October
1991; accepted
in final
form
28 April
1992.
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RI056
NASAL
STIMULATION
REFERENCES 1. Brill, R. W., and D. R. Jones. On the suitability of Innovar, a for cardiovascular experiments. Can. J. neuroleptic analgesic, Physiol. Pharmacol. 59: 1184-1189, 1981. 2. Daly, M. de B. Breath-hold diving: mechanisms of cardiovascular adjustments in the mammal. In: Recent Advances in Physiology, edited by P. F. Baker. New York: Churchill-Livingstone, 1984, p. 201-246. 3. Daly, M. de B., R. Elsner, and J. E. Angell-James. Cardiorespiratory control by carotid chemoreceptors during experimental dives in seals. Am. J. Physiol. 232 (Heart Circ. Physiol. 1): H508-H516, 1977. 4. Drummond, P. C., and D. R. Jones. The initiation and maintenance of bradycardia in a diving mammal, the muskrat, Ondatra zibethicus. J. Physiol. Lond. 290: 253-271, 1979. 5. Elsner, R., J. E. Angell-James, and M. de B. Daly. Carotid body chemoreceptor reflexes and their interactions in the seal. Am. J. Physiol. 232 (Heart Circ. Physiol. 1): H517-H525, 1977. 6. Huang, T. F., and Y. I. Peng. Role of the chemoreceptor in diving bradycardia in rat. Jpn. J. Physiol. 26: 395-401, 1976.
IN RATS 7. Kirk, R. E. Experimental Design: Procedures for the Behavioral Sciences. Belmont, CA: Wadsworth, 1982. 8. Lahiri, S., and R. G. DeLaney. Relationship between carotid chemoreceptor activity and ventilation in the cat. Resp. Physiol. 24: 267-286, 1975. 9. McCulloch, P. F., and D. R. Jones. Cortical influences on diving bradycardia in muskrats (Ondatra xibethicus). Physiol. 2001. 63: 1098-1117, 1990. 10. McRitchie, R. J., and S. W. White. Role of trigeminal, olfactory, carotid sinus and aortic nerves in the respiratory and circulatory responses to nasal stimulation of cigarette smoke and other irritants in the rabbit. Aust. J. Exp. Biol. Med. Sci. 52: 127-140, 1974. 11. Vatner, S. F., and E. B. Braunwald. Cardiovascular control mechanisms in the conscious state. N. Engl. J. Med. 293: 970-976, 1975. 12. West, N. H., and B. N. Van Vliet. Factors influencing the onset and maintenance of bradycardia in mink. Physiol. 2001. 59: 45l463, 1986. 13. Zar, J. H. Biostatistical Analysis. Englewood Cliffs, NJ: PrenticeHall, 1984.
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$2.00
Copyright
ulation, as during hypoxia or hypercapnia. A third objective was to reevaluate the importance of laryngeal receptor stimulation in generating the response. The cardiovascular responses to diving were simulated by nasal water flow with concurrent apnea in anesthetized, paralyzed, and artificially ventilated rats. Additionally, the cardiovascular effects of apnea were separated from those of nasal water flow in normoxic rats. METHODS Experiments were performed on male Sprague-Dawley rats (370-589 g). The procedures were approved by the Animal Care Committee of the University of Saskatchewan and conform to the standards of the Canadian Council on Animal Care. Surgical
Preparation
Rats were anesthetized with Innovar (Innovar-Vet, MTC Pharmaceuticals, Cambridge, Ontario; 0.15-0.2 ml/kg im, diluted to a 10% solution), after initial inhalation induction with methoxyflurane (Metofane, MTC Pharmaceuticals). Half-dose injections of Innovar were given hourly to maintain anesthesia. A femoral artery was cannulated (PE-50, Clay Adams, Parsippany, NJ) for arterial blood pressure measurement and arterial blood sampling. The ipsilateral femoral vein was cannulated for injection of a paralytic agent. Copper leads were inserted under the skin to record electrocardiogram (ECG), and a cardiotachometer (type 9857B, Beckman Instruments, Schiller Park, IL) was used to monitor heart rate. Body temperature was maintained at 38 k 1°C with a heating pad (Harvard Animal Temperature Control Unit, Ealing Scientific, St. Laurent, Quebec). The rats were placed in a supine position, the trachea was exposed through a ventral incision, and oral- and caudal-facing tracheal cannulas (PE-205, 1.57 mm ID) were inserted. The rats were then paralyzed with d-tubocurarine (Sigma, St. Louis, MO; 2 mg/kg iv), and ventilated through the caudal-facing cannula [model CTP-930, CWE, Ardmore, PA; respiratory fretidal volume (V,) adjusted for body wt; quency = 70 min-l, range 2.6-3.8 ml]. The oral-facing cannula was positioned with its tip just caudal to the epiglottis. The previously retracted submaxillary glands were repositioned, and the skin incision was tightly closed with wound clips (Autoclip, Clay Adams). In those trials involving water stimulation of the nasal region, room temperature tap water was pumped through the oral-facing cannula at 10 ml/min (model 50lU/R, Watson-Marlowe, Falmouth, England). If the water exited through the mouth and not through the external nares, the submaxillary glands and oral-facing cannula were repositioned, the incision was reclosed, and the trial was repeated. Experimental
Procedures
Nasal water flow trials with concurrent expiratory apnea (initiated by turning off the ventilator) were performed initially with the recurrent laryngeal nerves (RLN) and superior laryngeal nerves (SLN) intact. The water flow through the oralfacing cannula could not directly stimulate the receptors with afferents in the RLN and SLN because the cannula tip extended rostra1 to the larynx. However, stimulation of the laryngeal
0 1992 the American
Physiological
Society
R1049
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R1050
NASAL
STIMULATION
nerves was possible as water flow through the cannula could produce mechanical disturbances to stimulate the larynx. To eliminate possible laryngeal receptor stimulation, the RLN and SLN were sectioned bilaterally, and the nasal water flow with apnea trials were repeated. To investigate possible interactions between the cardiovascular effects of stimulation of the receptors in the nasal region and chemoreceptor drive, rats were ventilated to produce four groups with differing partial pressures of arterial 0, (Pao ) and CO, (Pace,). Arterial blood gases for the four groups were: >roup 1, Pao = ~80 mmHg and Pace = ~35 mmHg (normoxic and normocapnic condition); group 2”, Pao > 100 mmHg and Pace < 30 mmHg (hyperoxic and hypocapnic condition, designed to reduce peripheral and central chemoreceptor drive); group 3, Pao > 100 mmHg and Pace > 50 mmHg (hyperoxic and hypercapnic condition, designed to stimulate the chemoreceptors through an increased Pace,); and group 4, Pao, < 40 mmHg and Pace < 30 mmHg (hypoxic and hypocapnic condition, designed to stimulate the peripheral chemoreceptors through a lowered Pao,). Inspired gases were mixed with a gas mixing pump (Digamix, model M/300 A, Wosthoff, Bochum, Germany). Arterial pH, PO,, and PCO~ were monitored every 15-30 min (IL Micro 213 pH/blood gas analyzer, Instrumentation Laboratory, Lexington, MA), and V, and inspired fractions of N2, 02, and CO, were adjusted to ensure that blood gas values were appropriate for each group (Table 1). To separate the cardiac effects of apnea alone from those of nasal water flow during nasal water flow plus concurrent apnea, three experimental stimuli were used: apnea was expiratory apnea (initiated by turning off the ventilator), nasal water flow was nasal water flow through the oral-facing cannula without concurrent apnea, and nasal water flow plus concurrent apnea consisted of simultaneous nasal water flow and expiratory apnea. Six rats were used in each group, with each rat receiving three or more trials of each stimuli. All trials lasted 10 s. Five minutes separated each trial, and any water in the oral-facing cannula and nasal passages was cleared by blowing air through the cannula at the end of each trial. ECGs and arterial pressures were recorded on a Beckman R511A chart recorder.
IN RATS
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Means for each animal were calculated by determining average heart rate (HR) and mean arterial blood pressure (MABP) from all trials. Instantaneous HR was calculated from cardiac intervals, and MABP was calculated from pulsatile blood pressure traces (diastolic plus one-third pulse pressure). Grand means for each experimental stimulus were calculated by averaging the means from all animals. Values reported in text and in Figs. 2 and 4-8 are grand means * SE, with HR in beats per minute and MABP in millimeters Hg. Control HR and MABP were calculated by averaging three pretrial scores (at -5, -2.5, and 0 s) immediately preceding the onset of the experimental stimuli for each animal. Control HR and MABP between groups 1, 3, and 4 were found to be significantly different from each other (Table 2). To facilitate comparisons between these groups,
I
~-F
Fig. 1. Original chart recordings of nasal water flow plus concomitant expiratory apnea before (A) and after (B) the recurrent (RLN) and superior laryngeal nerves (SLN) were transected. Top to bottom: electrocardiogram (ECG), heart rate (HR), pulsatile arterial blood pressure (BP), ventilation (up, inspiration; down, expiration), event marker (down, onset of nasal water flow).
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2.5
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7.5
10.0
(s)
Fig. 2. HR (A) and mean arterial blood pressure (MABP) (B) responses (&SE) to nasal water flow plus expiratory apnea (n = 6) before and after RLN and SLN were cut. At no time were responses significantly different from each other. BPM, beats per minute.
Table 1. Arterial pH, Po2, and Pco2 values for four groups ventilated with different gases Group
1
2 3 4
PK
7.580t0.010" 7.646kO.021" 7.355k0.020h 7.551t0.011"
Pa0,,
mmHg
76.822.8" 106.3t2.6d l17.6k6Jd 35.9t1.2c
Pwo,,
mm&
34.lk1.3" 28.4kO.7' 55.8k2.6" 25.2tl.0f
Values are means t SE; n = 6 for each group. Groups: I, normoxic-normocapnic; 2, hyperoxic-hypocapnic; 3, hyperoxic-hypercapnic; hypoxic-hypocapnic. Value significantly different from: a arterial pH (pH,) values of groups 2 and 3; b all other pH, values; c all other arterial (Pao,) values; d Pao, values of groups 1 and 4; e all other arterial PCO~ (Paoo,) values; or f Pace, values of groups 1 and 3.
4, PO,
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NASAL
Table 2. Control
heart rate and mean arterial
STIMULATION
blood pressure
Apnea
Nasal
Rl051
IN RATS
for groups Water
1, 3, and 4 Nasal
Flow
Water
Flow
Plus
Apnea
Group HR
1 3 4
468.2t3.2* 393.2t2.7 364.8t2.1
MABP
88.7t0.3 88.3t0.3 46.8+0.1-f
HR
MABP
454.0t3.9” 384.7t5.9 370.3t7.4
91.9tO.l 92.1t0.3 46.4+0.5-f
Values are means t SE; n = 6 for each group. Groups: 1, normoxic-normocapnic; rate (HR) and mean arterial blood pressure (MABP) are beats/min and mmHg, and nasal water flow plus apnea, control HR and MABP were not significantly 2 groups, t MABP significantly lower than other 2 groups.
raw scores were transformed into a percent of control. This transformation did not affect the normality of the data distribution (13). Statistical analyses were performed with a computer package (SYSTAT, Systat, Evanston, IL). Student’s t test and one- and two-way analysis of variance (ANOVA) with repeated measures were employed, as required (7, 13). Statistical significance was reached if P was < 0.05. In the case of significant F values, Tukey’s honestly significant difference or Fisher’s least significant difference a posteriori tests were performed to determine significant differences among group means.
Nasal water flow plus concurrent apnea produced a rapid and substantial drop in HR and a rapid decrease in MABP (Fig. 1A). With the RLN and SLN intact, there was a 76.7% drop in HR (462.3 t 4.7 to 107.8 t 14.0 beats/min at 2.5 s) and a 41.1% decrease in MABP (89.7 t 0.3 to 52.8 t 4.4 mmHg) (Fig. 2). These responseswere not observed if the water exited through the oral cavity and not the external nares. The cardiovascular responses to nasal water flow plus apnea did not differ after the RLN and SLN were cut (Fig. 1B). HR decreased by 80.1% (451.0 t 5.8 to 89.9 t 7.5 beats/min at 2.5 s), and MABP decreased by 36.8% (89.9 t 0.3 to 56.8 t 4.9 mmHg at 2.5 s) (Fig. 2). This indicates that stimulation of afferents in the RLN and SLN is not required for elicitation of the cardiovascular responses to nasal water flow.
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360
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3, hyperoxic-hypercapnic; 4, hypoxic-hypocapnic. Units of heart respectively. Across experimental stimuli of apnea, nasal water flow, different within each group. * HR significantly greater than other
In the normoxic-normocapnic condition, nasal water flow plus apnea was more effective in inducing cardiovascular changes than was either of the two stimuli applied individually (Fig. 3). Throughout the duration of the trials, HR from the three stimuli were statistically different (Fig. 4). HR decreased the most during nasal water flow plus apnea, from 457.0 t 0.8 to 101.9 t 15.3 beats/min at 2.5 s. In comparison, HR decreased from 454.0 t 3.9 to 250.0 t 47.8 b eat s/ min at 2.5 s during nasal water flow, but only from 468.2 t 3.2 to 446.6 t 4.3 during apnea. During both nasal water flow and nasal water flow plus apnea, HR increased after the initial fall, whereas during apnea there was a slight decrease in HR throughout the duration of the trials. MABP for all three conditions was statistically different within the first 5 s, but MABP during nasal water flow then increased to a level not significantly different from that during apnea (Fig. 4). Throughout the duration of the trials, nasal water flow plus apnea MABP remained lower than control MABP. Apnea MABP increased above control; nasal water flow MABP initially decreased but then rose above control.
Cardiovascular Effects of Nasal Water Flow Plus Apnea
A
Role of Chemoreceptor Drive During Nasal Stimulation Withdrawal of chemoreceptor drive. The results from group 2 (hyperoxic-hypocapnic) are similar to group 1 (normoxic-normocapnic) (Fig. 5). During nasal water flow plus apnea, HR decreased immediately from 475.7 t 6.3 to 93.8 t 11.9 beats/min at 2.5 s and remained at that
B
C
1
a Fig. 3. Original chart recordings of apnea (A), nasal water flow (B), and nasal water flow plus apnea (C) in a normoxic-normocapnic rat. Apnea resulted in minimal HR and BP changes. Nasal water flow alone resulted in immediate but transient bradycardia and hypotension. Nasal water flow plus apnea resulted in an immediate bradycardia and hypotension that was sustained for duration of stimulus. Top to bottom: ECG, HR, pulsatile arterial BP, ventilation (up, inspiration; down, expiration), event marker (down, onset of stimulus).
'c; 100
2% mg
50 0I
VENTILATION EVENT MARKER
MABP
91.4tl.O 87.7t0.2 47.3+0.3-F
Importance of Concurrent Apnea During Nasal Stimulation
RESULTS
ECG
HR
457.0t0.8* 389.3t4.2 364.5t9.5
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Rl052
NASAL
STIMULATION
IN RATS
A
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Apnea
0
Nasal
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flow
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Fig. 4. HR (A) and MABP (B) responses (GE) to apnea, nasal water flow, and nasal water flow plus apnea for group 1 (normoxic-normocapnit) (n = 6). Nasal water flow plus apnea resulted in the greatest changes in HR and MABP, and apnea resulted in minimal changes. * Response significantly different from other 2 responses at that time; ** response significantly different from both apnea and nasal water flow responses at that time.
level for the duration of the stimulus. MABP decreased from 97.4 t 1.1 to 68.8 t 5.7 mmHg and remained at -75% of control MABP. With apnea, HR again showed little change while MABP increased above control levels. During nasal water flow, HR fell from 481.7 t 7.0 to 209.0 t 41.4 beats/min at 2.5 s but then increased to 358.4 t 39.6 beats/min at 7.5 s. MABP again initially decreased but then rose above control. HRs for the three experimental stimuli were statistically different from each other throughout the duration of the trials. Nasal water flow plus apnea MABP was significantly less than the other two stimuli. Chemoreceptor stimulation through preexisting hypercapnia or hypoxia. The cardiovascular responses to apnea, nasal water flow, and nasal water flow plus apnea for group 3 (hyperoxic-hypercapnic) and group 4 (hypoxichypocapnic) were compared with the responses for group 1 (normoxic-normocapnic). Control HR and MABP values for the three groups are presented in Table 2. Within each group, control HR and MABP were not significantly different across the three experimental stimuli. However, control HR for the three experimental stimuli was signif-
80
m
60
.-y iii 5
40
k s
20 O
CONTROL
2.5
5.0
Time
7.5
10.0
(s)
Fig. 5. HR (A) and MABP (B) responses (GE) to apnea, nasal water flow, and nasal water flow plus apnea for group 2 (hyperoxic-hypocapnit) (n = 6). Nasal water flow plus apnea resulted in the greatest changes in HR and MABP, and apnea resulted in minimal changes. HR and BP responses to nasal water flow alone showed an initial decrease but then became attenuated throughout duration of stimulus. * Response significantly different from other 2 responses at that time; ** response significantly different from both apnea and nasal water flow responses at that time.
icantly higher for group 1 than for groups 3 and 4. Control MABP was significantly lower for group 4 than for the other two conditions. Because of these differences in control values, HR and MABP were standardized to facilitate comparisons between groups. The cardiovascular responses to apnea, nasal water flow, and nasal water flow plus apnea for the three groups are presented in Figs. 6-8. HR remained constant and MABP increased above 100% in groups 1 and 3 during apnea (Fig. 6). In group 4, HR fell steadily to reach 36% of control at 10 s. MABP initially increased, but by 10 s MABP had decreased to 84%. During nasal water flow alone HR fell to -55% in all three groups by 2.5 s (Fig. 7). HR then increased and remained at -80% for the duration of the water flow. MABP initially decreased, but by 10 s all were above 100%. No response from the three groups was significantly different from any other during nasal water flow. During nasal water flow plus apnea (Fig.
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NASAL
V
Group
1
Group
3
Group
4
STIMULATION
IN
RATS
R1053
Fig. 6. Percent (GE) of control HR (A) and MABP (B) during apnea in group 1 (normoxic-normocapnic), group 3 (hyperoxic-hypercapnic), and group 4 (hypoxic-hypocapnit) (n = 6 for each group). Values are percent of control presented in Table 2. Group 4 showed a progressive decrease in HR and a decrease in MABP toward end of apneic period. * Response significantly different from the other 2 groups at that time; ** response significantly different from group 1 at that time.
t
2 0
60
$
50
I
CONTROL
I
I
I
2.5
5.0
7.5
Time
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1
10.0
(s)
8), HR decreased to 20% at 2.5 s in all three groups and rose slightly but remained at a decreased level (~45%) for the remainder of the stimulus duration. MABP fell to -67% in all three groups but then rose to -80%. Group 4 MABP decreased by a lesser extent than the other two groups, but only at 5 s were the differences between groups significant. DISCUSSION
Huang and Peng (6) reported that bilateral section of the carotid sinus nerve or destruction of the carotid bodies by intrasinusal injection of acetic acid in the conscious rat eliminated the bradycardic response to simulated diving. They concluded that the peripheral chemoreceptors were important in the manifestation of the cardiovascular responses to diving in the rat. This conclusion is surprising, as others have found that the carotid body chemoreceptors appear to be of little significance in initiation of the bradycardic response in both aquatic (muskrat, mink, and seal) and terrestrial mammals (rabbit) (3-5, 10, 12). The main conclusion of this study is that peripheral chemoreceptor stimulation is unnecessary for the initiation of the cardiovascular responses to simulated diving in the rat. This contradicts the findings of Huang and
Peng (6) but is in accordance with other mammalian literature regarding the role of chemoreceptor drive in breath-hold diving (3-5, 12). The main evidence for this conclusion is that after the chemoreceptors were offloaded during hyperoxic-hypocapnic ventilation, the cardiovascular responses to apnea, nasal water flow, and nasal water flow plus apnea were similar to those observed during the normoxic-normocapnic condition. Increased Pao or decreased Pace reduces peripheral chemoreceptor activity (8), thus functionally denervating the peripheral chemoreceptors. This method is preferable to other methods of reducing chemoreceptor input because all chemoreceptive sites, including carotid and aortic bodies and central chemoreceptors, become simultaneously and reversibly off-loaded. Attenuated chemoreceptor drive thus does not alter the cardiovascular responses to nasal stimulation observed during normal chemoreceptor drive. Further support for our conclusion that chemoreceptors do not play a role in the initial dive response of the rat is provided by cardiovascular responses to nasal water flow plus apnea during hyperoxic-hypercapnic and hypoxic-hypocapnic ventilation. If chemoreceptor drive is important for the manifestation of the diving response in
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R1054
t
NASAL
STIMULATION
IN RATS
100 0
’
80
2 i 0
60
Fig. 7. Percent (GE) of control HR (A) and MABP (B) during nasal water flow in group 1 (normoxic-normocapnit), group 3 (hyperoxic-hypercapnic), and group 4 (hypoxic-hypocapnic) (n = 6 for each group). Values are percent of control presen ted in Table 2. Responses were not significantly different from each other at any time. ;
100 0
4
90
T
2 0 0 y0
80 70
CONTROL
2.5
5.0 Time (s)
7.5
the rat, then increased chemoreceptor drive resulting from preexisting hypoxia or hypercapnia (8) should potentiate the cardiovascular responses to nasal water flow plus apnea. Our results indicate that this is not the case. The cardiovascular responses to nasal water flow plus apnea during normal chemoreceptor drive were no different from the responses observed when there was an increased chemoreceptor drive due to preexisting hypoxia or hypercapnia. Therefore, chemoreceptor stimulation through preexisting hypoxia or hypercapnia does not alter the cardiovascular responses to nasal water flow plus apnea. Preexisting hypoxic chemoreceptor drive did, however, alter the cardiac response to expiratory apnea. During hypoxic hypocapnia, HR fell progressively to 36% of its control, whereas HR in the normoxic and hypercapnic conditions changed only slightly. Slowly developing bradycardia during lung deflation in normoxic mink is partially alleviated by hyperoxia, suggesting an influence from the peripheral chemoreceptors (12). Carotid body stimulation contributes to the bradycardia toward the end of long-duration simulated dives in seals, helping to maintain the bradycardia during the developing asphyxia (3). Together these results indicate that chemoreceptor stimulation, either through progressive or preexisting hy-
10.0
poxia, results in a slowly developing bradycardia. Upon closer examination, the results of Huang and Peng (6) appear to be more of a response to progressive hypoxia than to upper airway stimulation. In the present study the short duration of the stimulus presumably precluded the development of substantial progressive hypoxia; thus only during preexisting hypoxia did the hypoxic chemoreceptor drive result in a slowly developing bradycardia during expiratory apnea. A further finding is that the rapid and intense bradycardia that develops during nasal water flow with concomitant apnea in anesthetized, paralyzed, and artificially ventilated rats is not due to stimulation of the recurrent or superior laryngeal nerves. The bradycardic response was unaltered after sectioning of these nerves. We concluded that the response appears to be due to stimulation of the trigeminal nerves innervating the nasal mucosa because no cardiovascular response was elicited when the water exited through the oral cavity rather than the external nares. Placing the animal in a supine position facilitated the water flow through the nares, and it was very important to clear as much water as possible from the nasal passages between trials to prevent a diminution of the response with repeated trials. The cardiovascular responses to nasal water flow plus
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NASAL
-t:
STIMULATION
IN
R1055
RATS
80
0
$
60
Fig. 8. Percent (&SE) of control HR (A) and MABP (B) during nasal water flow plus apnea in group 1 (normoxicnormocapnic), group 3 (hyperoxic-hypercapnic), and group 4 (hypoxic-hypocapnic) (n = 6 for each group). Values are percent of control presented in Table 2. * Response was significantly different from group 3 at that time.
CONTROL
2.5
5.0
Time
7.5
10.0
(s)
apnea included both bradycardia and hypotension. After laryngeal nerves were cut (Fig. Z), HR decreased by 80.1%, but MABP decreased by only 36.8%. If one assumes no change in stroke volume, decreased HR would cause cardiac output to fall, which would lead to a decreased MABP. However, the MABP decrease was only one-half that of the HR decrease. This suggests that nasal water flow plus apnea results in both a bradycardia and a peripheral vasoconstriction. A point worth addressing is the validity of comparing the cardiovascular responses of an anesthe tized, paralyzed, and ventilated preparation with those of the conscious animal (6). It has been shown that input from the cerebral cortex in conscious muskrats influences the magnitude of diving bradycardia by ~20% (9). Furthermore, although anesthesia can affect central interactions between afferent receptor groups (1 I), Innovar has been shown to have only mod&ate effects on cardiovascular function and reflexes (1). At least two species of mammals, apart from the rat, exhibit a similar rapid-onset bradycardia during both conscious diving (voluntary or involuntary submersion ) and nasal water flow when anesthetized, paralyzed, and ventilated (4 12) 9suggesting that the same receptor-driven response is initiated in both cases.
In summary, chemoreceptor stimulation does not play a major role in the initiation of diving bradycardia in the rat. Reduction of chemoreceptor stimulation, or chemoreceptor stimulation through preexisting hypoxia or hypercapnia, did not alter the cardiovascular responses to trigeminal stimulation by nasal water flow. This refutes the conclusions of Huang and Peng (6) but is consistent with other work in mammals. We found concomitant expiratory apnea during nasal stimulation necessary to sustain the substantial cardiovascular changes, although nasal stimulation without concurrent apnea still causes a large initial bradycardia and hypotension. Nasal water flow stimulates trigeminal receptors in the upper respiratory tract, and the-physiological responses to this stimulus were not due to activation of receptors with afferent fibers in the superior or recurrent laryngeal nerves. We thank Dr. Mark Evered for critical comments on the manuscript. This work was supported by grants from the Medical Research Council of Canada to N. H. West, and by Heart and Stroke Foundation of Saskatchewan Traineeships and a University of Saskatchewan Graduate Scholarship to P. F. McCulloch. Address for reprint requests: P. McCulloch, Dept. of Physiology, College of Medicine, Univ. of Saskatchewan, Saskatoon, Saskatchewan S7N OWO, Canada. Received
21 October
1991; accepted
in final
form
28 April
1992.
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RI056
NASAL
STIMULATION
REFERENCES 1. Brill, R. W., and D. R. Jones. On the suitability of Innovar, a for cardiovascular experiments. Can. J. neuroleptic analgesic, Physiol. Pharmacol. 59: 1184-1189, 1981. 2. Daly, M. de B. Breath-hold diving: mechanisms of cardiovascular adjustments in the mammal. In: Recent Advances in Physiology, edited by P. F. Baker. New York: Churchill-Livingstone, 1984, p. 201-246. 3. Daly, M. de B., R. Elsner, and J. E. Angell-James. Cardiorespiratory control by carotid chemoreceptors during experimental dives in seals. Am. J. Physiol. 232 (Heart Circ. Physiol. 1): H508-H516, 1977. 4. Drummond, P. C., and D. R. Jones. The initiation and maintenance of bradycardia in a diving mammal, the muskrat, Ondatra zibethicus. J. Physiol. Lond. 290: 253-271, 1979. 5. Elsner, R., J. E. Angell-James, and M. de B. Daly. Carotid body chemoreceptor reflexes and their interactions in the seal. Am. J. Physiol. 232 (Heart Circ. Physiol. 1): H517-H525, 1977. 6. Huang, T. F., and Y. I. Peng. Role of the chemoreceptor in diving bradycardia in rat. Jpn. J. Physiol. 26: 395-401, 1976.
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