Abstract Experiments were performed on unanesthetized rats (n=6) to determine the systemic hemodynamics during chemoreflex activation by intravenous KCN. Rats chronically instrumented with ultrasonic flow probes in the ascendant aorta were submitted to KCN injections (30 μg/kg) before and after sequential administration of the autonomic blockers atropine and propranolol. In the control period KCN injections produced a 60% reduction in heart rate (HR) and a 46% elevation in blood pressure (BP), while cardiac output (CO) decreased 76%, stroke volume (SV) decreased 40%, and calculated total peripheral resistance (TPR) increased 900%. Atropine administration increased resting HR, whereas no change was observed in CO or BP. Chemoreflex-induced bradycardia was markedly attenuated (26%), and the pressor response was potentiated (59%) after atropine administration. CO and TPR responses were both attenuated after atropine administration (68% and 718%, respectively). Sequential administration of propranolol decreased HR but did not change the cardiovascular responses to KCN injections compared with the responses observed after atropine administration. In conclusion, CO is greatly reduced during KCN-evoked chemoreflex. Besides the intense bradycardia, a decrease in SV contributed to this reduction. Bradycardic response was most dependent on the cardiac parasympathetic activation, and the reduction in SV was probably most dependent on the increased cardiac afterload due to the sudden increase in BP.
In addition to respiratory responses, stimulation of the peripheral chemoreceptors evokes cardiovascular responses that result from complex interactions of (1) primary activation of sympathetic and parasympathetic neurons by the chemoreceptor input, (2) the modulation of this neural activation by the simultaneous hyperventilation and activation of cardiopulmonary and arterial baroreceptors, and (3) changes in arterial blood gases that may affect direct or indirectly the cardiovascular and neural functions.1 2 Each of these mechanisms is more or less expressive depending on the characteristics of the stimulus used to activate carotid body chemoreceptors and on the species used. The primary neural mechanisms prevail when the secondary modulation by hyperventilation and cardiovascular reflexes are prevented. In this case, bradycardia, due to cardiac parasympathetic activation, and increases in peripheral resistance, due to vascular sympathetic activation, dominate the cardiovascular responses. The secondary modulation influences both the cardiac and vascular responses. The accumulated evidence indicates that the activation of pulmonary stretch receptors during hyperventilation induces tachycardia, due to cardiac vagal inhibition and cardiac sympathetic stimulation, and reduces the peripheral resistance, due to inhibitory influence on the vascular sympathetic neurons.1 2 This modulation has been shown to be more important and dominates the cardiovascular responses to chemoreceptor stimulation if the ventilation increases more than 200% from baseline during chemoreflex activation.3 4 In the rat, a species in which the resting respiratory frequency is elevated, peripheral chemoreceptor stimulation does not augment ventilation by more than 100% from baseline.5 This is probably the cause for the minor modulation of cardiovascular responses by hyperventilation in this species.6
The type, intensity, and duration of the stimulus used to activate the chemoreflex also affects the final cardiovascular responses. The stimulus per se may affect directly other neural or local vascular and cardiac mechanisms. These effects may dominate or mask the final cardiovascular responses to chemoreceptor stimulation. For example, hypoxemia, the natural stimulus, in addition to the direct activation of the carotid body chemoreceptors, influences the vascular and cardiac muscle contractility, favoring vasodilatation and reduction in cardiac output. Depending on the intensity and duration of the hypoxia, the effects on vascular and cardiac muscles may dominate the vascular response over the sympathetic activation inducing vasodilatation and decrease in arterial pressure.7 8 9
In previous studies,5 10 we reported the cardiovascular and ventilatory responses to chemoreflex activation by intravenous injections of KCN. This maneuver eliminates some of the difficulties imposed by the multiplicity of actions of the natural peripheral chemoreceptor stimulants (hypoxia and hypercapnia), especially the direct cardiac and vascular influences. Moreover, this allows the study of cardiovascular responses to chemoreceptor stimulation in unanesthetized rats, an animal too small for the necessary instrumentation for direct stimulation of peripheral chemoreceptors in chronic preparations. Typically, intravenous KCN produces, in the first 10 seconds following the injection, a dose-related bradycardia, pressor responses, tachypnea, and alerting. These responses almost completely disappear after carotid body chemoreceptor elimination. Experiments performed with autonomic blockers indicated that the marked bradycardia was almost exclusively related to cardiac parasympathetic activation and the pressor responses to vascular sympathetic activation.
In our previous studies the role of neural mechanisms in the cardiovascular responses to chemoreflex activation by KCN was clarified in some extension. However, the cardiovascular parameters used to assess the cardiovascular responses (heart rate and blood pressure) did not allow a more complete description of the hemodynamics and mechanisms involved in the cardiovascular responses to chemoreflex activation by this maneuver. To study the response of the systemic hemodynamic parameters to chemoreflex evoked by KCN, rats were chronically instrumented with transit-time ultrasonic flow probes in the ascendant aorta, along with arterial and venous catheters, and KCN injections were performed. The effect of the cardiac parasympathetic activation on the cardiac output responses was obtained by comparing the responses before and after atropine administration. To assess the influence of the cardiac sympathetic effects on the cardiac output responses to KCN, injection of this drug was performed after propranolol administration.
Experiments were performed on six adult male Wistar rats (260 to 330 g) obtained from animal facilities of the School of Medicine, University of São Paulo (São Paulo, Brazil). The rats were housed in the animal facilities center at the Heart Institute, School of Medicine, University of São Paulo with food and water provided ad libitum, before the experimental protocol. All procedures followed the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, Md 20892] and the guidelines of the Animal Welfare Act.
For instrumentation the rats were anesthetized with a mixture of ketamine (100 mg/kg IM) and diazepam (1.5 mg/kg IM) and placed on a heated surgical table to maintain body temperature at 37°C. The trachea was cannulated with a Gelco tube and the ventilation controlled with a Harvard ventilator for small animals (model 683). Tygon and polyvinyl–tipped cannulas were placed in the femoral artery and vein, tunneled subcutaneously, and exteriorized at the back of the neck. Transonics 2SB flow probes were implanted in the ascending thoracic aorta at the same surgical time as the vascular catheterization throughout a thoracotomy performed at the third right intercostal space. These surgical procedures were performed under aseptic conditions, and in the recovery period all animals received a single intravenous dose of penicillin G benzathine (Benzetacil, Fontoura-Wyeth), 60 000 U. Animals were allowed to recover for 5 days before study.
Pulsatile arterial pressure and cardiac output were monitored continuously during the experiments. The arterial catheter was connected to a COBE transducer (Arvada), and the signal was amplified with a GP4A-General Purpouse Amplifier (Stemtech). The ultrasonic volume flowsensor was connected to a T206 Transonic flowmeter. The amplifier and flowmeter outputs were connected to an A/D board and this to a computer loaded with a CODAS Data Acquisition software (AT-CODAS; DATAQ Instruments). Blood pressure and cardiac output were continuously recorded at a sample rate of 100 Hz each. Stroke volume was calculated from the following formula: Stroke Volume=Cardiac Output/Heart Rate. Total peripheral resistance was calculated from the following formula: Total Peripheral Resistance=Mean Arterial Pressure/Cardiac Output. Heart rate was obtained from the average of the intervals between two systolic peaks in pulsatile blood pressure recordings.
Chemoreflex was activated by intravenous injections of KCN, as described previously.5 KCN solution in distilled water was prepared fresh every day, protected from the light with aluminum foil, and injected intravenously in a dose of 30 μg/kg in volumes of 0.15 to 0.0 mL per injection. The analysis of the cardiovascular responses to KCN injection for individual rats was performed as follows. Baseline blood pressure, cardiac output, and heart rate were taken immediately prior to the KCN injection. Changes in blood pressure were determined by the difference of the baseline values and the maximum increase, while changes in cardiac output and heart rate were determined by the difference of baseline values and the maximum decrease following KCN injection. In some animals a brisk fall in blood pressure preceded the pressor responses, as indicated in the example of Fig 1a⇓. In the animals in which this phenomena occurred this was not considered for the analysis of the pressor responses to KCN.
Pulsatile blood pressure and cardiac output were monitored continuously for a variable control period (≈30 minutes) until the animals remained quiet in a Plexiglas cage and the systemic hemodynamic variables were stable. The experiments were repeated in two consecutive days in the same animals. After hemodynamic stability was reached, three separate injections of a single dose of KCN were performed in the animals. This was done because the cardiovascular responses to KCN are somewhat variable. The injections were separated by a 10 minute period or until the cardiovascular parameters had returned to control values. After this section, atropine (4 mg/kg) was administered intravenously and again 10 minutes after the KCN injections were repeated. Sequentially, propranolol (4 mg/kg) was administered intravenously and again 10 minutes after the KCN injections were repeated. The doses of atropine and propranolol were tested in a separate group of rats for the effectiveness of the autonomic blockade. The experiments were repeated in two consecutive days in the same animals, and the data of each day were averaged to give a single value per rat. The data of systemic hemodynamics for the three KCN injections under each condition (ie, before and after autonomic blockade) were averaged, and the values obtained for each day were again averaged to produce a single value per rat.
Data are presented as mean±SEM. Differences among mean values between different treatments were tested with one-way ANOVA. Duncan’s multiple-range test was used as a post hoc analysis if the probability from the F test was <.05.
During the control period mean arterial pressure averaged 116±2.5 mm Hg, heart rate 407±9.5 bpm, cardiac output 83±3.2 mL/min, cardiac index 31.4±0.02 mL/min/100 g body wt, calculated stroke volume 0.21±0.01 mL, and calculated total peripheral resistance 1.5±0.08 PRU (n=6). Fig 1⇑ shows an example of the effect of KCN injections on blood pressure, heart rate, and cardiac output in one rat. Typically, KCN injections produced a marked increase in blood pressure and a marked decrease in heart rate. In the control period the average increase of blood pressure was 53±3 mm Hg (46%), while heart rate decreased −245±22 bpm (60%) (Fig 2⇓). These responses were accompanied by a 76% decrease in cardiac output (−64±4 mL/min) and a 40% decrease in calculated systolic volume (−0.08±0.017 mL). The calculated total peripheral resistance increased 14±2.24 PRU.
Effect of Autonomic Blockade on Hemodynamic Responses
Administration of atropine increased resting heart rate to 424±6 bpm. Mean arterial pressure (112±2.9 mm Hg), cardiac output (89±3.6 mL/min), and the calculated stroke volume (0.21±0.012) did not change significantly, compared with values before atropine administration. Fig 1b⇑ shows the cardiovascular response to KCN after atropine administration in the same rat shown in Fig 1a⇑. Atropine clearly attenuated the bradycardia to chemoreflex that was evoked by KCN. The average responses are demonstrated in Fig 2⇑. Heart rate decreased by −109±29 bpm (26%), while the responses of cardiac output (−55±6.6 mL/min, 62%) and stroke volume (0,1±0.015 mL, 48%) were not different from the responses observed in the control period. The pressor response was potentiated by atropine (66±3.8 mm Hg, 59%) and the calculated total peripheral resistance increased 9.5±2.2.
The sequential administration of propranolol decreased heart rate to 390±11 bpm, while mean arterial pressure (127±2 mm Hg), cardiac output (72±3.7), calculated stroke volume (0.19±0.01 mL), and total peripheral resistance (1.8±0.09 PRU) did not change significantly, compared with the same parameters after atropine administration (Fig 1c⇑). In response to KCN, heart rate decreased −158 bpm (40%) and blood pressure increased 58±5 mm Hg (46%) after propranolol administration. The cardiac output decreased 67% (−47±7.6 mL/min) and calculated stroke volume decreased 44% (−0.08±0.01 mL). These differences were not statistically significant compared with the responses observed after atropine administration.
The results of the present study extended previous data from our laboratory concerning cardiovascular responses to chemoreflex activation by intravenous injections of KCN. In previous studies,5 10 we demonstrated that most of the cardiovascular, respiratory, and behavioral responses to intravenous KCN are related to the stimulation of carotid body chemoreceptors, the only functionally active peripheral chemoreceptor contingent in adult rats. Injections of KCN in rats lacking the carotid body chemoreceptors produced only slight changes in blood pressure, heart rate, and ventilation. In addition, the typical behavioral response (alerting/defense response) was no longer observed after elimination of carotid body chemoreceptors. These data support the assumption that the systemic hemodynamic responses to KCN observed in the present study were also related to chemoreflex activation.
In addition to the previously reported bradycardia and pressor response, the present data demonstrated that the chemoreflex activation evoked by intravenous KCN in unanesthetized rats produces a marked decrease in cardiac output and an increase in total peripheral resistance. The decrease in cardiac output was due to a simultaneous decrease in heart rate and in stroke volume. Bradycardia in response to KCN is mostly determined by the cardiac parasympathetic activation. This was indicated in the present study and in previous studies by the marked attenuation in the heart rate decrease after atropine administration. This is probably uniquely related to a direct parasympathetic activation by the chemoreceptors’ afferent fibers. Simultaneous activation of cardiovascular mechanoreceptors could also be claimed to contribute to this response. However, some characteristics of the response appear to negate a role for the mechanoreceptors’ influence. Activation of mechanoreceptors produces changes in heart rate of lower magnitude than those observed with KCN. The actual increase in pressure (as seen in Fig 1a⇑), occurs after the maximum fall in heart rate or cardiac output. Thus, it seems unlikely that the increase in blood pressure, which occurs later, would be responsible for activating the cardiac or arterial baroreceptors to produce the effects observed. Finally, in previous studies the elimination of aortic baroreceptors (the most important baroreceptor contingent in the rat), did not change the magnitude of the heart rate responses to KCN. The decrease in stroke volume also contributed to the observed decrease in cardiac output during KCN-induced chemoreflex. Our present data indicate that the decrease in stroke volume during chemoreflex activation by KCN may be related to the augmented cardiac afterload produced by the sudden rise in blood pressure.
Different from data obtained with local stimulation of carotid body chemoreceptors in anesthetized rats2 6 and in unanesthetized rats subjected to systemic hypoxia,7 9 the data from the present study indicated that the chemoreflex activation by KCN in unanesthetized rats produces a marked increase in peripheral resistance. As we demonstrated before, this response is mediated by a direct sympathetic activation by the chemoreflex.5 The dominance of the sympathetic influence on the vascular responses to chemoreflex activation evoked by KCN in unanesthetized rats may be related to at least four different factors. First, the less important modulation of the sympathetic activity by the hyperventilation-induced stretch receptors’ activation in the rat compared with dogs allows the full manifestation of the direct sympathetic activation by the chemoreceptor afferent fibers.3 4 9 Second, the absence of important direct vascular effects of KCN in the dose used and in the time frame in which the responses were observed in the present study (first 10 seconds). This contrasts with results obtained in unanesthetized animals10 subjected to systemic hypoxia. In this case, local influences dominate the vascular responses over the sympathetic activation by the hypoxia-induced chemoreflex. Third, chemoreflex and baroreflex can mutually interact. Experimental evidences obtained in various species showed that during simultaneous activation of both reflexes, the chemoreceptor afferent input may attenuate the baroreflex ability to modulate sympathetic activity.1 2 11 12 The inhibition of the baroreflex action during chemoreflex activation by KCN could enhance the sympathetic activity.12 Finally, chemoreflex activation by KCN in conscious rats evokes behavioral responses similar to those observed in the defense reaction.5 This could reinforce the increase in sympathetic activity both directly by primary sympathetic activation and indirectly by attenuating the buffering function of the baroreflex. In fact, the last one is a well known effect of the stimulation of the defense areas.2 12 This factor could not be investigated in the studies performed under anesthetics because they prevent afferent activation of the defense areas.
In summary, the present study demonstrated that the cardiovascular responses to chemoreflex stimulation by KCN are characterized by a simultaneous bradycardia and pressor response, decreased cardiac output, decreased stroke volume, and an increase in peripheral vascular resistance. The substantial decrease in cardiac output is related to combined bradycardia and the decrease in stroke volume. The stroke volume decreases seen during chemoreflex activation evoked by KCN are probably more related to the great increase in the cardiac afterload produced by the sudden increase in blood pressure. On the other hand, bradycardia is related to cardiac parasympathetic activation by the chemoreceptor afferent fibers. The great increase in vascular resistance in response to chemoreflex activated by KCN is probably related to multiple influences that lead to an increase in sympathetic activity. This includes direct activation by the peripheral chemoreceptors, activation of the defense areas, and inhibition of the baroreflex action. Moreover, in the rat, the lack of an important influence of the sympathetic inhibitory action of the pulmonary stretch reflex probably contributes to the full manifestation of the increased sympathetic activity during chemoreflex activation by KCN.
- Received March 17, 1997.
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