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The effects of carotid body hypocapnia on ventilation in goats
Respiration Physiology, 79 (1990) 123-136
123
Elsevier
RESP 01623
The effects of carotid body hypocapnia on ventilation in goats Leighann Daristotle, Anne D. Berssenbrugge, Michael J. Engwall and Gerald E. Bisgard Departments of Comparative Biosciences and Veterinary Science, University of Wisconsin, Madison, Wisconsin 53706, U.S.A. (Accepted for publication 2 November 1989) Abstract. This study was designed to examine the influence of carotid body (CB) hypocapnia on ventilation by selectively perfusing the CB through an extracorporeal circuit in 19 goats. When Pcbco z was decreased from normocapnic levels in 14 awake goats (APcbcoz = 10.9 Torr), Paco 2 increased 5.6 Torr (P < 0.05) and "~/Edecreased 24 ~o (P < 0.00 l) (mean values). The ventilatory sensitivity to inspired CO 2 was not changed by CB hypocapnia in 5 of these goats, but the response was shifted to the right. During CB hypocapnia, ventilatory instability, including apnea, was observed in 4 of 14 goats; this irregular breathing continued at elevated levels of Paco 2. In 5 anesthetized goats, CB hypocapnia (APcbco~ = 18.0 Torr) decreased VE by 70~o in the intact state, but produced no significant ventilatory depression after CB denervation. We conclude that CB hypocapnia depresses ventilation in both awake and anesthetized goats mostly through CB chemoreceptor effects, and suggest that this hypoventilation may predispose to ventilatory instability in some animals.
Hypocapnia; Carotid body; Control of breathing; CO2 response; Goats
The carotid body (CB) has been known to be a CO2 sensor as well as an 02 sensor since the work of Heymans et al. in 1930. Studies have demonstrated increases in carotid sinus nerve (CSN) afferent activity in response to increases in Paco 2 (Samaan and Stella, 1935; Hornbein etal., 1961; Fitzgerald and Parks, 1971; Lahiri and Delaney, 1975), and corresponding increases in ventilation in response to CB hypercapnia (Fitzgerald et al., 1964; Heeringa et al., 1979). Hypocapnia, on the other hand, has been shown to reduce CSN afferent traffic (Samaan and Stella, 1935), and hypocapnic CB perfusates depressed ventilation in anesthetized dogs (Heymans et al., 1930; Fitzgerald et al., 1964). Because our awake goat CB perfusion model allows us to restrict a hypocapnic stimulus to the CB and enables us to examine the response to this stimulus in the absence of anesthesia, we wished to characterize the ventilatory effects of selective CB hypocapnia using this model. Correspondence address: L. Daristotle, Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706, U.S.A. 0034-5687/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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Methods
General. Nineteen adult goats were studied while undergoing CB perfusion; the animals were divided into 3 experimental groups. Group 1 consisted of 7 awake goats in which the ventilatory response to CB hypocapnia was characterized. In Group 2, we examined the effects of CB hypocapnia before and after CB denervation in 5 anesthetized goats. In Group 3, we observed the response to CB hypocapnia and assessed the interaction of CB hypocapnia and elevated systemic Pa¢o 2 in 7 awake goats. Carotid body perfusion model. The carotid body perfusion model has been described in detail previously (Busch et al., 1985), and a brief summary follows. The anatomical characteristics of cerebral blood flow in the goat provided a basis for surgical separation of brain and CB perfusion. Brain blood supply in this species is derived from the bilateral internal maxillary arteries via the rete mirabile; the contribution of the vertebral artery is negligible. Ligation of one carotid artery at its bifurcation into internal maxillary and lingual arteries prevents the blood perfusing the ipsilateral carotid body from also perfusing the brain. Rearrangement of the vasculature to allow CB perfusion with blood of independent gas tension and pH from that perfusing the CNS required 2 separate surgeries under general anesthesia in each of the goats studied. The first phase of surgical preparation for isolated CB perfusion included unilateral ligation of the internal maxillary and lingual arteries on the CB perfusion side and, contralaterally, excision of the CB and ligation of the occipital artery on the brain perfusion side. After a minimum 2 weeks of recovery the second phase was completed, which included insertion ofpolyvinyl arterial sampling catheters and placement of silastic cannulae in the distal common carotid artery on the CB side and in the right atrium via the ipsilateral jugular vein. An arteriovenous shunt was formed by connecting these cannulae, so that between studies blood flowed from the aorta through the contralateral carotid artery into the anastomosing ipsilateral occipital artery and into the short segment of carotid artery remaining in the region of the CB, and was returned via the externalized shunt to the right heart. Sodium heparin (20 000-40 000 units, subcutaneously) was administered each day after shunt implementation. Extracorporeal perfusion circuit. This circuit drew blood from the goat's right heart through the venous cannula into a venous reservoir (RV-500-1, SciMed Life Systems) by means of one head of a 2-headed perfusion pump (Travenol). Blood was pumped from the reservoir by the other pump head into a heat exchanger (P-714, SciMed Life Systems) kept near body temperature, and through a membrane oxygenator (0400-2A or 1500-2A, SciMed Life Systems), blood filter (LPE 1440, Pall) and into the CB perfusion cannula. Perfusion blood gas tensions were effected by changing the relative concentrations of CO2, 02, and N 2 flowing through the gas phase of the oxygenator. Perfusion pressure was measured using a 'T' connector located just proximal to the CB cannula and connected to a pressure transducer. Perfusion pressure was maintained 15-40 Torr above systemic arterial pressure during experiments.
VENTILATION IN CAROTID BODY HYPOCAPNIA
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Experimental studies. Awake goats were studied no sooner than 5 days after the second surgery. They stood quietly in a stanchion during the experiments and breathed through a muzzle mask. Anesthetized goats were studied the day of their second surgery. They were intubated orally with an endotracheal tube following intravenous administration of sodium thiamylal (Surital) and placed in supine position on a table. Anesthesia was maintained during placement of perfusion cannulae and femoral artery and vein catheters, and during experimental protocols, with intravenous chloralose (10-15 mg/kg/h). Intravenous NaHCO 3 and Lactated Ringers Solution were given to maintain normal acid-base balance and prevent hypotension. All goats breathed through a unidirectional Hans Rudolph valve, attached to the endotracheal tube of anesthetized goats and to the facemask of awake goats. The expiratory side of the valve was connected to a respirometer, and the inspiratory side was connected'to a gas mixing chamber. A pneumotachograph was positioned in the expiratory tubing in Group 1 and 2 studies, and in the inspiratory tubing in Group 3 studies. A CO2 analyzer monitored end-tidal CO2. Systemic arterial blood pressure, CB perfusion pressure, expired Pco2, inspiratory or expiratory flow and respirometer potentiometer output were recorded continuously on a 6-channel polygraph. Blood samples were drawn anaerobically into heparinized syringes and immediately analyzed for pH, Pco2 and Po2 (Radiometer, Copenhagen). Blood gas tensions were corrected using values obtained from fresh goat blood equilibrated with known gas mixtures in a tonometer. Standard methods were used to correct values for temperature (Severinghaus, 1966). Prowcols: Groups 1 and 2. Following a control period of normocapnic-normoxic CB perfusion with normal systemic blood gases, Pcbco: was decreased as much as possible while Pcbo2 was kept normoxic. Supplemental oxygen was sometimes added to the inspired gas to maintain systemic normoxia. Systemic arterial Pco2 was determined by the level of pulmonary ventilation. Systemic arterial and CB perfusion blood samples were obtained every 3-5 min, and ventilation was measured continuously during data collection periods. Exposure to CB hypocapnic perfusion lasted 10-30 min; goats were subjected to 1-2 exposures per study. Hypocapnic CB perfusion was repeated in anesthetized goats (Group 2) after denervation of the perfused CB by surgical ligation of the carotid sinus nerve. Denervation was confirmed by lack of hyperventilation following bolus injection of 1 mg NaCN through the CB perfusion cannula. Protocol: Group 3. Following a control period ofnormocapnic-normoxic CB perfusion with the animal breathing room air, systemic CO2 ventilatory response curves in systemic hyperoxia were performed in each goat during CB perfusion with normocapnic-normoxic blood and hypocapnic-normoxic blood. This protocol yielded 2 systemic (non-CB) CO2 responses at different steady-state levels of CB stimulation. Curves were accomplished by stepwise increases in the fraction of CO 2 in the inspiratory mixing chamber. Systemic arterial and CB perfusion blood gases were drawn and ventilation
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was measured after 5 minutes at each level of FIco2. Responses were done in random order, and multiple responses with the same CB blood conditions were completed in some animals. The ventilatory response to CB hypocapnia was determined in Group 3 goats through measurements of blood gases and ventilation after 5 minutes of CB hypocapnia, prior to increasing Fi¢o ~. Ventilation was analyzed for ~'E, f and VT for all studies; in addition, TI and TE were measured in Group 3 experiments. All volumes were corrected to BTPS. Multiple responses performed on the same day in the same goat were averaged to give one mean response for each awake animal, and a mean response before and after CB denervation in each anesthetized animal. Mean control ~'E, VT, f, Pcbco ~ and P a c o ~ for all awake goats were compared to the means of measurements made in CB hypocapnia through Student's t-test. A P-value of 0.05 or less was considered indicative of statistical significance in all statistical comparisons. AVE/APcbco2, an evaluation of CB CO 2 sensitivity, was also calculated as the change in ventilation due to a change in Pcbco 2 from hypocapnic to normocapnic levels. The same comparisons were made in anesthetized goats between CB normocapnic and CB hypocapnic values. In addition, A'¢E/APcbco ~ was compared before and after CB denervation by paired t-test. To evaluate the breath-to-breath variability of VT, TI and TE in Group 3 goats, the coefficients of variation were calculated for these ventilatory measurements as the standard deviations divided by the mean values from 20 consecutive breaths in CB normocapnic-normoxia and CB hypocapnia. Responses to CO z were estimated through linear regression of VE, VT and f on systemic P a c o 2. The slopes and x-intercepts of responses done in CB normocapnic normoxia were compared with those performed in CB hypocapnia through paired t-tests. Data analysis.
Results
Carotid body hypocapnia resulted in significant increases in P a c o 2 and significant decreases in minute ventilation and tidal volume in all groups of goats studied (table 1). This hypoventilation was stable within 5 min of a decrease in the F c o 2 of the gas supplying the oxygenator. The 10.9-Torr decrease in mean Pcbco 2 diminished "¢E by 24~o and VT by 17~o, and Paco 2 subsequently rose 15~o in Group 1 and 3 goats. In addition, CB hypocapnia abolished spontaneous ventilation in 3 of the 5 anesthetized goats from Group 2 (fig. 1). The other 2 anesthetized goats decreased "V'E,VT and f b y 41 ~o, 24~o and 25~o in response to the 18.1-Torr decrease in mean (Group 2) Pcbco2. Mean Paco~ rose 24~o in Group 2 animals. Carotid sinus nerve section eliminated the hypoventilatory effects of CB hypocapnia in Group 2 goats (table 1). No significant change in ventilation, P a c o 2, frequency and tidal volume was seen during carotid body hypocapnia after denervation. In addition,
45.1 ± 2.2 27.0 ± 2.6*
49.7 ± 2.5 24.9 _+ 1.4"
Anesthetized (Group 2) CB Intact Control CB hypocapnia
CB denervated Control CB hypocapnia 51.2 ± 1.6 50.6 ± 1.9
46.2 ± 2.7 57.4 + 2.4*
36.9 ± 1.1 42.5 + 1.3"
Paco 2 (Torr)
* Significantly different than corresponding control (P < 0.05). ** Significantly different than CB intact. + + Apnea occurred in 3 of 5 goats. Value is average of remaining 2.
37.2 + 0.7 26.3 ± 1.3"
Awake (Groups 1 and 3) Control CB hypocapnia
Pcb¢o 2 (Torr) 1)
5.96 ± 0.62 5.34 + 0.65
6.37 ± 0.72 3.76 -'+
9.49 + 0.72 7.25 + 0.78*
"¢E (L. min -
21.6 ± 1.7 21.2 + 2.3
21.3 ± 2.9 16 ++
18.0 ± 1.3 16.5 ± 1.5
f (br" min l)
0.28 ± 0.03 0.25 ± 0.02
0.31 ± 0.03 0.24 ÷÷
0.53 ± 0.03 0.44 ± 0.03*
VT (L)
0.03 _+ 0.01"*
0.27 + 0.09
0.29 + 0.07
A~'E/APcbco2 (L" min - 1. T o r r - ' )
Changes in Pcbco2, Paco2, ~tE and AVE/APcbco 2in awake and anesthetized goats subjected to selective carotid body per±us±on. Pcbo2 = 108.9 ± 2.6 Torr in control and 130.1 _+7.7 Torr in CB hypocapnia; Pao2 = 122.8 ± 11.0 Torr in control and 113.9 _+ 13.0 Torr in CB hypocapnia. All values are means ± S.E.
TABLE 1
7
Z
0 >
0
0
>
>
Z
<
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L. DARISTOTLE et al. CB HYPOCAPNIA
CONTROL
50
(Torr)
PETco2
,
0 200 Arterial BP
(Torr) 0 200 I (Torr) 0"5[0[
'"0[
CB perfuslon P
[ VT
JJ JJJJJJ JJJJJJJLJJJ JJJJ uJ 30 s
Fig. 1. Record from an anesthetized goat during CB normocapnia (control) and CB hypocapnia. Apnea occurred after 5 min exposure to CB hypocapnic perfusion and lasted 2.1 min.
AVE/APcbco 2, the ventilatory sensitivity to decreases in Pcbco2, was nearly abolished by CB denervation. The hypoventilation in CB hypocapnia was manifest as marked ventilatory instability, including apnea, in 4 of the 14 awake goats of Groups 1 and 3. Tidal volume and the duration of inspiration and expiration showed increased variability in CB hypocapnia (fig. 2). For example, the mean coefficient of variation of VT, TI and TE calculated for 2 goats from Group 3 was elevated from 11.1 ~o in CB normocapnia to 31.1 ~o in CB hypocapnia; in the other goats from this group, these coefficients did not significantly change with decreases in Pcbco ~. This increased breath-to-breath variation resulted in large oscillations in the level of ventilation in 2 goats from Group 1 and 2 goats from Group 3. Mean Pa¢o: increased 8.2 Torr in these 4 animals within 5 min of CB hypocapnia, compared with 3.7 Torr in the other awake goats. One of the Group 3 goats exhibited ventilatory instability and apneic periods of 10-50 sec during its first exposure
A. C O N T R O L VT0"5 r (I)
oL
20 0.5
/
B. CB HYPOCAPNIA
~
$ *
Fig. 2. Tidal volume tracings showing a marked increase in the breath-to-breath variability of ventilatory volume and timing during CB hypocapnia in this awake goat. Pcb¢o2 in A. = 36.7 Torr; in B., 23.4 Torr.
129
VENTILATION IN CAROTID BODY HYPOCAPNIA TABLE 2
Mean CB blood gases and slopes and Paco2-intercepts for responses to changes in Paco 2 in Group 3 (n = 7) goats. Mean Pao2 = 147.1 + 11.7 Torr. Values are + S.E.
Pcbco2, Torr Pcbo2 , Torr
Normocapnic-normoxic CB
Hypocapnic CB
38.2 104.9
27.2 133.9
+ 0.4 + 1.8
+ 2.3 _+ 12.6
Slope ~rE, L" min ~.Torr 1 VT, L ' T o r r ] f, b r . m i n J . T o r r i
2.285 + 0.467 0.045 + 0.007 1.085 _ 0.241
2.538 + 0.609 0.060 + 0.013 1.135 _+ 0.260
Paco2-intercept "QE, Torr VT, Torr f, Torr
30.14 _+ 1.34 16.85 _ 4.29 17.78 _+ 3.99
38.60 _+ 2.38* 30.61 _+ 1.59" 26.43 + 4.64
* Significantly different from normocapnic-normoxic value at P < 0.05.
to CB hypocapnia, associated with an initial 11-Torr increase in Paco 2. A second exposure 2 h later resulted in simple hypoventilation and increased Paco 2 only 4.3 Torr. Carotid body hypocapnia had no effect on the ventilatory sensitivity to Paco ~ in 5 of the 7 Group 3 goats. In these animals, lowering the Pcbco~. 11 Torr did not significantly change the slope of the systemic CO2 response for VE, VT and f, but significantly increased the Paco2-intercept values for "¢E and VT (table 2). Thus, the non-CB chemoreceptor response to COz was not blunted by CB hypocapnia, but was shifted to the right. During periods of unstable breathing in CB hypocapnia, the effective ventilatory sensitivity to systemic CO: was greatly diminished. In 2 of the 7 Group 3 goats studied, Pcbco 2 : 3 7 . 6
Torr
Pcb¢o 2
31.8
Torr
Pa¢o 2 = 6 8 . 1 T o r r
Paco 2 = 4 1 . 9 T o r r VT 0.5L
[
30 s
Fig. 3. Ventilatory record from one goat during CB normocapnia and during CB hypocapnia with increased FIc¢ 2. Breathing remained unstable in CB hypocapnia when Paco 2 was elevated.
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L. DARISTOTLE et al.
CB hypocapnia could produce apneas or irregular ventilatory patterns, and this instability persisted as P a c o 2 was increased (fig. 3). These measurements of breathing were therefore not well-correlated (r 2 < 0.70) with P a c o 2 during CB hypocapnia. In one of these 2 goats, a second exposure to CB hypocapnia later in the experiment resulted in a stable hypoventilation and a brisk P a c o 2 response.
Discussion Perfusion of the vascularly isolated CB with hypocapnic blood resulted in significant hypoventilation in awake and anesthetized goats, an effect which was abolished by CB denervation in anesthetized goats. This hypoventilation was associated with a CB hypocapnia-induced shift of the systemic (central chemoreceptor) response toward higher Paco2S in most awake goats studied, and with ventilatory instability in some awake goats. Critique of methods. Separation of CB and brain blood flows is a characteristic of our CB perfusion model which was essential in these studies. We are confident that central neural structures were not exposed to the blood gas conditions delivered to the CB. Validation studies have been reported previously (Busch et al., 1985) in which no evidence of significant mixing between blood perfusing the CB and that perfusing the brain was detected. In addition, we injected radiopaque dye into the CB perfusion cannula in one of the animals used in these experiments and radiographic angiography confirmed that the CB circulation did not include brain vasculature. The aortic bodies were intact in our preparation and were exposed to the systemic circulation; thus, they potentially could have contributed to the ventilatory responses to elevated P a c o 2 in these experiments. We minimized aortic chemoreceptor effects during the CO z responses by maintaining systemic hyperoxia. We also tested for extra-CB peripheral chemosensitivity by intravenous administration of NaCN (50 #g/kg) that was restricted from the CB circulation by the perfusion circuit; no hyperventilatory response to NaCN was found in these animals, indicating absence of significant extra-CB peripheral chemoreflexes. Thus, we feel that aortic chemoreceptor effects are not important in our results. We used simple linear regression to estimate the slopes of the ventilatory responses to CO2 in this study. Although VT and f may not be truly linear functions of P a c o 2, the mean coefficients of determination (r 2) for VE, VT and f were greater than 0.70 in 5 of 7 Group 3 goats. We conclude that these simple linear models were an adequate fit for our data and provided useful information about these responses. CB hypocapnia and hypoventilation. Carotid body hypocapnia produced a hypoventilatory response in goats similar to that observed by Heymans et al. (1930) and Fitzgerald et aL (1964) during perfusion of carotid chemoreceptors with hypocapnic blood in anesthetized dogs. Our unanesthetized goats exhibited a 24~o depression of VE from
VENTILATION IN CAROTID BODY HYPOCAPNIA
131
control during CB hypocapnia, comparable to the 24~o decrease seen in the dogs of Fitzgerald et al. (1964). The major effect of CB hypocapnia on ventilation in goats was a reduction of tidal volume. This is compatible with evidence that the ventilatory response to changes in Paco 2 is mostly due to changes in tidal volume or phrenic amplitude (Davis and Stagg, 1975; Speck and Webber, 1985). In addition, data on awake hypercapnic men exposed suddenly to transient hypocapnia revealed that the resulting peripheral chemoreceptormediated decrease in ventilation was produced more by decreases in tidal volume than by decreases in respiratory rate (Miller et al., 1974). The hypoventilation demonstrated during selective perfusion of the CB with hypocapnic blood was most likely a carotid chemoreceptor effect. Carotid chemodenervation abolished the ventilatory consequences of CB hypocapnia in our anesthetized goats and in anesthetized dogs (Fitzgerald et al., 1964). In addition, single chemoreceptor unit discharge frequency recordings during CO 2 response curves obtained in our laboratory from anesthetized goats suggest that ventilatory drive from CB chemoreceptor input is very low at the levels of Pcbco: achieved in this study (Engwall et al., 1988). In 12 single fibers, plots of discharge frequency vs. Paco2 show a mean Paco2-intercept of 25.1 Tort at 0 imp/sec, approximating the Pco: values producing ventilatory depression in CB-perfused goats. Although actual discharge rates did not reach zero, the mean discharge rate of 5 fibers studied in the hypocapnic range was 15~ of the mean normocapnic discharge rate (0.22 imp/sec at Paco2 = 20.1-28.0 Ton" vs. 1.48 imp/sec at Paco ~ = 40 Torr). Carotid sinus nerve activity was also found to be much reduced or eliminated by systemic hypocapnia in anesthetized cats (Samaan and Stella, 1935). It is interesting to note that CB denervation resulted in a less marked depression of ventilation than did CB hypocapnia (table 1). One might expect that removal of CB chemoreceptor input by denervation would produce at least equivalent ventilatory effects as the decreased CB chemoreceptor afferent traffic associated with CB hypocapnia. One possible explanation for this disparity would be the ventilatory effects of elimination of baroreceptor input in sectioning the carotid sinus nerve. Ventilation has been shown to increase with decreasing carotid sinus baroreceptor stimulation via decreasing isolated sinus pressure in anesthetized dogs (Brunner et al., 1982). Interruption of carotid sinus baroreceptor afferents in our CB denervations might likewise stimulate breathing and counteract the depressant effect of loss of CB chemoreceptor drive. Perfusion of the neurally intact CB, however, should have little effect on baroreceptor function, and the baroreceptor contribution to the ventilatory response to CB hypocapnia should be negligible. The degree of hypoventilation observed during CB hypocapnia varied considerably among goats. This response was not dependent upon the magnitude of change of PcbcQ (fig. 4); the animals exhibiting the largest decreases in ventilation were not those subjected to the largest falls in Pcbco ~. The rate of change and the 'shape' of the decline in Pco~ at the CB chemoreceptor have been shown to affect ventilation (Dutton et al., 1967), but we cannot address the importance of these factors as we lack this information for our model.
132
L. DARISTOTLE et al. 0 • •
-25
•
co
-50 -75 -100 ~ o -20
t
i
-15
-10
A P°.o.,
i
-5 (Torr)
0
Fig. 4. Decreases in ventilation as percentages of control values produced by decreases in Pcbco 2in awake goats, Groups 1 and 3 (.) and anesthetized goats, Group 2 (o). Ventilatory depression did not appear to be related to the magnitude of CB hypocapnia in these animals. Note that 3 of the 5 anesthetized goats became apneic ( - 100~o A~'E)when APcbco2 = - 15 to -20 Torr. Peripheral chemosensitivity t o C O 2 may partially account for the trends seen in our data. Individual ventilatory responses to CB hypercapnia performed in 5 goats from Group 1 correlated well with responses to CB hypocapnia ( r 2 > 0.80); goats with greater depression of ventilation in CB hypocapnia tended to hyperventilate more in CB hypercapnia. This relationship suggests that ventilation may be a linear function of P c b c o 2 in the range examined in some goats. Central chemoreceptor responses will also determine the level of ventilation during CB hypocapnia. The dependence of the respiratory controller on peripheral vs. central chemoreceptor CO2 sensitivities may be important here, and may vary widely between animals (Heeringa et aL, 1979). Our data suggests that CB hypocapnia did not interact with central chemosensitivity in producing hypoventilation. In the majority of goats studied, CB hypocapnia had no significant effect on the slope of the extra-CB CO2 ventilatory response, but resulted in a shift of the response toward lower ventilatory and higher P a c o 2 values. We have previously shown in this same model that the ventilatory response to hypercapnic-hypoxic CB perfusion, like that from hypocapnic perfusion, is integrated with central chemosensitivity in an additive manner (Daristotle and Bisgard, 1987). The greater degree of ventilatory depression seen in anesthetized goats may have been, in part, the result of chloralose increasing the Pco2 at which apnea occurred (Florez and Borison, 1969). In this manner, CB hypocapnia of similar magnitude could elicit simple hypoventilation in an awake animal and apnea in a chloralose-anesthetized one. CB hypocapnia and ventilatory instability. Carotid body hypocapnia produced large oscillations in ventilatory timing and volume in a minority of Group 1 and 3 goats studied. Many factors have been proposed to affect respiratory stability, including
VENTILATION IN CAROTID BODY HYPOCAPNIA
133
chemosensitivity, conscious state, body gas stores, and circulatory effects (Cherniack and Longobardo, 1986). We suggest that the highly variable breathing patterns seen in CB hypocapnia could be due to a combination of systemic hypercapnia, central chemosensitivity, CB chemosensitivity and spontaneous ventilatory oscillations. Hypercapnia can contribute to ventilatory instability by increasing the effectiveness of ventilation in changing arterial blood gas tensions. When resting Paco 2 is elevated, a change in ventilation will produce a greater change in Paco 2because of the hyperbolic shape of the metabolic curve. This increase in the gain of the control of Paco 2 would increase the gain of the overall negative feedback control loop relating ventilation and Paco 2, and could result in system instability and oscillations in ventilation (Chapman et al., 1988). Carotid body hypocapnia decreased ventilation and increased Paco 2in our goats, and therefore could predispose to such oscillations. Indeed, the 4 goats with irregular breathing in CB hypocapnia also showed the greatest initial changes in Paco 2. In addition, ventilatory instability was associated with a larger increase in Paco ~ than was stable hypoventilation when one goat was subjected to CB hypocapnia twice. This systemic hypercapnia would be sensed chiefly through the central medullary chemoreceptor; the CB chemoreceptor was exposed only to steady-state hypocapnia. Central chemosensitivity would therefore dictate the response to the increase in Paco ~. An enhanced systemic CO 2 response could lead to respiratory oscillations by increasing the feedback loop gain. Although our data are insufficient to fully evaluate the role of central chemosensitivity in CB hypocapnic ventilatory instability, the (CB normocapnic-normoxic) systemic CO2 ventilatory response slope of one of the 4 goats was greater than the mean slope (2.79 vs. 2.29 L/min/Torr), and could have contributed to the increased variability of breathing during CB hypocapnia in this animal. Carotid body chemoreceptor function could promote unstable breathing in CB hypocapnia. In our experiments, the CB was exposed to steady-state normoxic hypocapnia, and could not respond to changes in blood gas tensions resulting from alterations of ventilation. Because stability of respiratory output is thought to be dependent on the controller's ability to be informed of the consequences of its actions (Cherniack and Longobardo, 1986), this effective lack of CB sensitivity to the chemical sequelae of ventilatory changes, combined with the reduction in tonic CB chemoreceptor input to the controller, may result in ventilatory instability during CB hypocapnia. We observed spontaneous cycles in the pattern of breathing during CB normocapnicnormoxia in one of the goats that developed ventilatory instability in CB hypocapnia. Such spontaneous oscillations have been observed more frequently in sleeping humans which developed periodic breathing in hypoxia than in those which did not (Chapman et al., 1988). These oscillations could be magnified when feedback loop gain is increased (by systemic hypercapnia in our goats or hypoxia in the sleeping humans), and then lead to marked ventilatory instability. It is possible that changes in the state of arousal could be involved in the irregular breathing observed in CB hypocapnia. Decreases in the level of consciousness might facilitate apnea and instability, and variation in alertness could explain, at least in part, the variation between goats and within experiments in the ventilatory effects of CB
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L. DARISTOTLE et al.
h y p o c a p n i a . H o w e v e r , w e did n o t e v a l u a t e c o n s c i o u s state in o u r g o a t s in t h e s e studies, a n d t h e r e f o r e c a n n o t s u p p o r t o r refute this theory. W e d o n o t feel it is likely that t r a n s i e n t c h a n g e s in C B p e r f u s a t e c o n d i t i o n s c o u l d be the s o u r c e o f the v e n t i l a t o r y instability. A l t h o u g h w e did n o t m a k e c o n t i n u o u s m e a s u r e m e n t o f C B b l o o d p H a n d gas t e n s i o n s , t h o s e v a l u e s w e o b t a i n e d w e r e stable during the h y p o c a p n i c trials, a n d c h a n g e s in v e n t i l a t i o n did n o t t r a c k small c h a n g e s in C B conditions.
Acknowledgements.The authors wish to thank Mr Gordon Johnson, Ms Margaret Rankin and Ms Kristine Bisgard for their valuable technical assistance. This work was supported by USPHS grants HL15473, HL07052 and HL07655.
References Brunner, M.J., M.S. Sussman, A.S. Greene, C.H. Kallman and A.A. Shoukas (1982). Carotid sinus baroreceptor reflex control o'f respiration. Circ. Res. 51: 624-636. Busch, M. A., G. E. Bisgard and H. V. Forster (1985). Ventilatory acclimatization to hypoxia is not dependent on arterial hypoxemia. J. AppL Physiol. 58: 1874-1880. Chapman, K.R., E.N. Bruce, B. Goethe and N.S. Cherniack (1988). Possible mechanisms of periodic breathing during sleep. J. Appl. Physiol. 64: 1000-1008. Cherniack, N.S. and G.S. Longobardo (1986). Abnormalities in respiratory rhythms. In: Handbook of Physiology. Section 3. The Respiratory System. Control of Breathing. Vol. II, edited by N. S. Cherniack and J.G. Widdicombe. Bethesda, MD, Am. Physiol. Soc., pp. 729-749. Daristotle, L. and G. E. Bisgard (1989). Central-peripheral chemoreceptor ventilatory interaction in awake goats. Respir. PhysioL 76: 383-392. Davis, J. N. and D. Stagg (1975). Interrelationships of the volume and time components of individual breaths in resting man. J. Physiol. (London) 24: 481-498. Dutton, R. E., W.A. Hodson, D.G. Davies and A. Fenner (1967). Effect of the rate of rise of carotid body Pco2 on the time course of ventilation. Respir. Physiol. 3: 367-379. EngwaU, M.J.A., E. H. Vidruk, A.M. Nielsen and G.E. Bisgard (1988). Response of the goat carotid body to acute and prolonged hypercapnia. Respir. Physiol. 74: 335-344. Fitzgerald, R. S., J.T. Zajtchuk, R. W. B. Penman and J. F. Perkins (1964). Ventilatory response to transient perfusion of carotid chemoreceptors. Am. J. Physiol. 207: 1305-1313. Fitzgerald, R.S. and D.C. Parks (1971). Effect of hypoxia on carotid chemoreceptor response to carbon dioxide in cats. Respir. Physiol. 12: 218-229. Florez, J. and H.L. Borison (1969). Effects of central depressant drugs on respiratory regulation in the decerebrate cat. Respir. Physiol. 6:318-329. Heeringa, J., A. Berkenbosch, J. De Goede and C.N. Olievier (1979). Relative contribution of central and peripheral chemoreceptors to the ventilatory response to CO 2 during hyperoxia. Respir. Physiol. 37: 365-379. Heymans, C., J.J. Bouckaert and L. Dautrebande (1930). Sinus carotidien et rdflexes respiratoires. II. Influences respiratoires r6flexes de racidose, de ralcalose, de ranhydride carbonique, de rion hydrog6ne et de ranox6mie. Sinus carotidiens et 6changes respiratoires dans les poumons et au-del~t des poumons. Arch. Int. Pharmacodynam. Ther. 39: 400-448. Hornbein, T.F., Z:J. Griffo and A. Roos (1961). Quantitation of chemoreceptor activity: interrelation of hypoxia and hypercapnia. J. Neurophysiol. 24: 561-568.
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Lahiri, S. and R.G. Delaney (1975). Stimulus interaction in the responses of carotid body chemoreceptor single afferent fibers. Respir. PhysioL 24: 249-266. Miller, J.P., D.J.C. Cunningham, B.B. Lloyd and J.M. Young (1974). The transient respiratory effects in man of sudden changes in alveolar CO 2 in hypoxia and high oxygen. Respir. Physiol. 20: 17-31. Samaan, A. and G. Stella (1935). The response of the chemical receptors of the carotid sinus to the tension of CO2 in the arterial blood in the cat. J. Physiol. (London) 85: 309-319. Severinghaus, J.W. (1966). Blood gas calculator. J. Appl. Physiol. 21 : 1108-1116. Speck, D. F. and C. L. Webber (1985). Effect of hypoxic, hyperoxic or hypercapnic ventilation on inspiratory termination in the cat. Respir. Physiol. 60: 205-215.
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The effects of carotid body hypocapnia on ventilation in goats Leighann Daristotle, Anne D. Berssenbrugge, Michael J. Engwall and Gerald E. Bisgard Departments of Comparative Biosciences and Veterinary Science, University of Wisconsin, Madison, Wisconsin 53706, U.S.A. (Accepted for publication 2 November 1989) Abstract. This study was designed to examine the influence of carotid body (CB) hypocapnia on ventilation by selectively perfusing the CB through an extracorporeal circuit in 19 goats. When Pcbco z was decreased from normocapnic levels in 14 awake goats (APcbcoz = 10.9 Torr), Paco 2 increased 5.6 Torr (P < 0.05) and "~/Edecreased 24 ~o (P < 0.00 l) (mean values). The ventilatory sensitivity to inspired CO 2 was not changed by CB hypocapnia in 5 of these goats, but the response was shifted to the right. During CB hypocapnia, ventilatory instability, including apnea, was observed in 4 of 14 goats; this irregular breathing continued at elevated levels of Paco 2. In 5 anesthetized goats, CB hypocapnia (APcbco~ = 18.0 Torr) decreased VE by 70~o in the intact state, but produced no significant ventilatory depression after CB denervation. We conclude that CB hypocapnia depresses ventilation in both awake and anesthetized goats mostly through CB chemoreceptor effects, and suggest that this hypoventilation may predispose to ventilatory instability in some animals.
Hypocapnia; Carotid body; Control of breathing; CO2 response; Goats
The carotid body (CB) has been known to be a CO2 sensor as well as an 02 sensor since the work of Heymans et al. in 1930. Studies have demonstrated increases in carotid sinus nerve (CSN) afferent activity in response to increases in Paco 2 (Samaan and Stella, 1935; Hornbein etal., 1961; Fitzgerald and Parks, 1971; Lahiri and Delaney, 1975), and corresponding increases in ventilation in response to CB hypercapnia (Fitzgerald et al., 1964; Heeringa et al., 1979). Hypocapnia, on the other hand, has been shown to reduce CSN afferent traffic (Samaan and Stella, 1935), and hypocapnic CB perfusates depressed ventilation in anesthetized dogs (Heymans et al., 1930; Fitzgerald et al., 1964). Because our awake goat CB perfusion model allows us to restrict a hypocapnic stimulus to the CB and enables us to examine the response to this stimulus in the absence of anesthesia, we wished to characterize the ventilatory effects of selective CB hypocapnia using this model. Correspondence address: L. Daristotle, Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706, U.S.A. 0034-5687/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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Methods
General. Nineteen adult goats were studied while undergoing CB perfusion; the animals were divided into 3 experimental groups. Group 1 consisted of 7 awake goats in which the ventilatory response to CB hypocapnia was characterized. In Group 2, we examined the effects of CB hypocapnia before and after CB denervation in 5 anesthetized goats. In Group 3, we observed the response to CB hypocapnia and assessed the interaction of CB hypocapnia and elevated systemic Pa¢o 2 in 7 awake goats. Carotid body perfusion model. The carotid body perfusion model has been described in detail previously (Busch et al., 1985), and a brief summary follows. The anatomical characteristics of cerebral blood flow in the goat provided a basis for surgical separation of brain and CB perfusion. Brain blood supply in this species is derived from the bilateral internal maxillary arteries via the rete mirabile; the contribution of the vertebral artery is negligible. Ligation of one carotid artery at its bifurcation into internal maxillary and lingual arteries prevents the blood perfusing the ipsilateral carotid body from also perfusing the brain. Rearrangement of the vasculature to allow CB perfusion with blood of independent gas tension and pH from that perfusing the CNS required 2 separate surgeries under general anesthesia in each of the goats studied. The first phase of surgical preparation for isolated CB perfusion included unilateral ligation of the internal maxillary and lingual arteries on the CB perfusion side and, contralaterally, excision of the CB and ligation of the occipital artery on the brain perfusion side. After a minimum 2 weeks of recovery the second phase was completed, which included insertion ofpolyvinyl arterial sampling catheters and placement of silastic cannulae in the distal common carotid artery on the CB side and in the right atrium via the ipsilateral jugular vein. An arteriovenous shunt was formed by connecting these cannulae, so that between studies blood flowed from the aorta through the contralateral carotid artery into the anastomosing ipsilateral occipital artery and into the short segment of carotid artery remaining in the region of the CB, and was returned via the externalized shunt to the right heart. Sodium heparin (20 000-40 000 units, subcutaneously) was administered each day after shunt implementation. Extracorporeal perfusion circuit. This circuit drew blood from the goat's right heart through the venous cannula into a venous reservoir (RV-500-1, SciMed Life Systems) by means of one head of a 2-headed perfusion pump (Travenol). Blood was pumped from the reservoir by the other pump head into a heat exchanger (P-714, SciMed Life Systems) kept near body temperature, and through a membrane oxygenator (0400-2A or 1500-2A, SciMed Life Systems), blood filter (LPE 1440, Pall) and into the CB perfusion cannula. Perfusion blood gas tensions were effected by changing the relative concentrations of CO2, 02, and N 2 flowing through the gas phase of the oxygenator. Perfusion pressure was measured using a 'T' connector located just proximal to the CB cannula and connected to a pressure transducer. Perfusion pressure was maintained 15-40 Torr above systemic arterial pressure during experiments.
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Experimental studies. Awake goats were studied no sooner than 5 days after the second surgery. They stood quietly in a stanchion during the experiments and breathed through a muzzle mask. Anesthetized goats were studied the day of their second surgery. They were intubated orally with an endotracheal tube following intravenous administration of sodium thiamylal (Surital) and placed in supine position on a table. Anesthesia was maintained during placement of perfusion cannulae and femoral artery and vein catheters, and during experimental protocols, with intravenous chloralose (10-15 mg/kg/h). Intravenous NaHCO 3 and Lactated Ringers Solution were given to maintain normal acid-base balance and prevent hypotension. All goats breathed through a unidirectional Hans Rudolph valve, attached to the endotracheal tube of anesthetized goats and to the facemask of awake goats. The expiratory side of the valve was connected to a respirometer, and the inspiratory side was connected'to a gas mixing chamber. A pneumotachograph was positioned in the expiratory tubing in Group 1 and 2 studies, and in the inspiratory tubing in Group 3 studies. A CO2 analyzer monitored end-tidal CO2. Systemic arterial blood pressure, CB perfusion pressure, expired Pco2, inspiratory or expiratory flow and respirometer potentiometer output were recorded continuously on a 6-channel polygraph. Blood samples were drawn anaerobically into heparinized syringes and immediately analyzed for pH, Pco2 and Po2 (Radiometer, Copenhagen). Blood gas tensions were corrected using values obtained from fresh goat blood equilibrated with known gas mixtures in a tonometer. Standard methods were used to correct values for temperature (Severinghaus, 1966). Prowcols: Groups 1 and 2. Following a control period of normocapnic-normoxic CB perfusion with normal systemic blood gases, Pcbco: was decreased as much as possible while Pcbo2 was kept normoxic. Supplemental oxygen was sometimes added to the inspired gas to maintain systemic normoxia. Systemic arterial Pco2 was determined by the level of pulmonary ventilation. Systemic arterial and CB perfusion blood samples were obtained every 3-5 min, and ventilation was measured continuously during data collection periods. Exposure to CB hypocapnic perfusion lasted 10-30 min; goats were subjected to 1-2 exposures per study. Hypocapnic CB perfusion was repeated in anesthetized goats (Group 2) after denervation of the perfused CB by surgical ligation of the carotid sinus nerve. Denervation was confirmed by lack of hyperventilation following bolus injection of 1 mg NaCN through the CB perfusion cannula. Protocol: Group 3. Following a control period ofnormocapnic-normoxic CB perfusion with the animal breathing room air, systemic CO2 ventilatory response curves in systemic hyperoxia were performed in each goat during CB perfusion with normocapnic-normoxic blood and hypocapnic-normoxic blood. This protocol yielded 2 systemic (non-CB) CO2 responses at different steady-state levels of CB stimulation. Curves were accomplished by stepwise increases in the fraction of CO 2 in the inspiratory mixing chamber. Systemic arterial and CB perfusion blood gases were drawn and ventilation
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was measured after 5 minutes at each level of FIco2. Responses were done in random order, and multiple responses with the same CB blood conditions were completed in some animals. The ventilatory response to CB hypocapnia was determined in Group 3 goats through measurements of blood gases and ventilation after 5 minutes of CB hypocapnia, prior to increasing Fi¢o ~. Ventilation was analyzed for ~'E, f and VT for all studies; in addition, TI and TE were measured in Group 3 experiments. All volumes were corrected to BTPS. Multiple responses performed on the same day in the same goat were averaged to give one mean response for each awake animal, and a mean response before and after CB denervation in each anesthetized animal. Mean control ~'E, VT, f, Pcbco ~ and P a c o ~ for all awake goats were compared to the means of measurements made in CB hypocapnia through Student's t-test. A P-value of 0.05 or less was considered indicative of statistical significance in all statistical comparisons. AVE/APcbco2, an evaluation of CB CO 2 sensitivity, was also calculated as the change in ventilation due to a change in Pcbco 2 from hypocapnic to normocapnic levels. The same comparisons were made in anesthetized goats between CB normocapnic and CB hypocapnic values. In addition, A'¢E/APcbco ~ was compared before and after CB denervation by paired t-test. To evaluate the breath-to-breath variability of VT, TI and TE in Group 3 goats, the coefficients of variation were calculated for these ventilatory measurements as the standard deviations divided by the mean values from 20 consecutive breaths in CB normocapnic-normoxia and CB hypocapnia. Responses to CO z were estimated through linear regression of VE, VT and f on systemic P a c o 2. The slopes and x-intercepts of responses done in CB normocapnic normoxia were compared with those performed in CB hypocapnia through paired t-tests. Data analysis.
Results
Carotid body hypocapnia resulted in significant increases in P a c o 2 and significant decreases in minute ventilation and tidal volume in all groups of goats studied (table 1). This hypoventilation was stable within 5 min of a decrease in the F c o 2 of the gas supplying the oxygenator. The 10.9-Torr decrease in mean Pcbco 2 diminished "¢E by 24~o and VT by 17~o, and Paco 2 subsequently rose 15~o in Group 1 and 3 goats. In addition, CB hypocapnia abolished spontaneous ventilation in 3 of the 5 anesthetized goats from Group 2 (fig. 1). The other 2 anesthetized goats decreased "V'E,VT and f b y 41 ~o, 24~o and 25~o in response to the 18.1-Torr decrease in mean (Group 2) Pcbco2. Mean Paco~ rose 24~o in Group 2 animals. Carotid sinus nerve section eliminated the hypoventilatory effects of CB hypocapnia in Group 2 goats (table 1). No significant change in ventilation, P a c o 2, frequency and tidal volume was seen during carotid body hypocapnia after denervation. In addition,
45.1 ± 2.2 27.0 ± 2.6*
49.7 ± 2.5 24.9 _+ 1.4"
Anesthetized (Group 2) CB Intact Control CB hypocapnia
CB denervated Control CB hypocapnia 51.2 ± 1.6 50.6 ± 1.9
46.2 ± 2.7 57.4 + 2.4*
36.9 ± 1.1 42.5 + 1.3"
Paco 2 (Torr)
* Significantly different than corresponding control (P < 0.05). ** Significantly different than CB intact. + + Apnea occurred in 3 of 5 goats. Value is average of remaining 2.
37.2 + 0.7 26.3 ± 1.3"
Awake (Groups 1 and 3) Control CB hypocapnia
Pcb¢o 2 (Torr) 1)
5.96 ± 0.62 5.34 + 0.65
6.37 ± 0.72 3.76 -'+
9.49 + 0.72 7.25 + 0.78*
"¢E (L. min -
21.6 ± 1.7 21.2 + 2.3
21.3 ± 2.9 16 ++
18.0 ± 1.3 16.5 ± 1.5
f (br" min l)
0.28 ± 0.03 0.25 ± 0.02
0.31 ± 0.03 0.24 ÷÷
0.53 ± 0.03 0.44 ± 0.03*
VT (L)
0.03 _+ 0.01"*
0.27 + 0.09
0.29 + 0.07
A~'E/APcbco2 (L" min - 1. T o r r - ' )
Changes in Pcbco2, Paco2, ~tE and AVE/APcbco 2in awake and anesthetized goats subjected to selective carotid body per±us±on. Pcbo2 = 108.9 ± 2.6 Torr in control and 130.1 _+7.7 Torr in CB hypocapnia; Pao2 = 122.8 ± 11.0 Torr in control and 113.9 _+ 13.0 Torr in CB hypocapnia. All values are means ± S.E.
TABLE 1
7
Z
0 >
0
0
>
>
Z
<
128
L. DARISTOTLE et al. CB HYPOCAPNIA
CONTROL
50
(Torr)
PETco2
,
0 200 Arterial BP
(Torr) 0 200 I (Torr) 0"5[0[
'"0[
CB perfuslon P
[ VT
JJ JJJJJJ JJJJJJJLJJJ JJJJ uJ 30 s
Fig. 1. Record from an anesthetized goat during CB normocapnia (control) and CB hypocapnia. Apnea occurred after 5 min exposure to CB hypocapnic perfusion and lasted 2.1 min.
AVE/APcbco 2, the ventilatory sensitivity to decreases in Pcbco2, was nearly abolished by CB denervation. The hypoventilation in CB hypocapnia was manifest as marked ventilatory instability, including apnea, in 4 of the 14 awake goats of Groups 1 and 3. Tidal volume and the duration of inspiration and expiration showed increased variability in CB hypocapnia (fig. 2). For example, the mean coefficient of variation of VT, TI and TE calculated for 2 goats from Group 3 was elevated from 11.1 ~o in CB normocapnia to 31.1 ~o in CB hypocapnia; in the other goats from this group, these coefficients did not significantly change with decreases in Pcbco ~. This increased breath-to-breath variation resulted in large oscillations in the level of ventilation in 2 goats from Group 1 and 2 goats from Group 3. Mean Pa¢o: increased 8.2 Torr in these 4 animals within 5 min of CB hypocapnia, compared with 3.7 Torr in the other awake goats. One of the Group 3 goats exhibited ventilatory instability and apneic periods of 10-50 sec during its first exposure
A. C O N T R O L VT0"5 r (I)
oL
20 0.5
/
B. CB HYPOCAPNIA
~
$ *
Fig. 2. Tidal volume tracings showing a marked increase in the breath-to-breath variability of ventilatory volume and timing during CB hypocapnia in this awake goat. Pcb¢o2 in A. = 36.7 Torr; in B., 23.4 Torr.
129
VENTILATION IN CAROTID BODY HYPOCAPNIA TABLE 2
Mean CB blood gases and slopes and Paco2-intercepts for responses to changes in Paco 2 in Group 3 (n = 7) goats. Mean Pao2 = 147.1 + 11.7 Torr. Values are + S.E.
Pcbco2, Torr Pcbo2 , Torr
Normocapnic-normoxic CB
Hypocapnic CB
38.2 104.9
27.2 133.9
+ 0.4 + 1.8
+ 2.3 _+ 12.6
Slope ~rE, L" min ~.Torr 1 VT, L ' T o r r ] f, b r . m i n J . T o r r i
2.285 + 0.467 0.045 + 0.007 1.085 _ 0.241
2.538 + 0.609 0.060 + 0.013 1.135 _+ 0.260
Paco2-intercept "QE, Torr VT, Torr f, Torr
30.14 _+ 1.34 16.85 _ 4.29 17.78 _+ 3.99
38.60 _+ 2.38* 30.61 _+ 1.59" 26.43 + 4.64
* Significantly different from normocapnic-normoxic value at P < 0.05.
to CB hypocapnia, associated with an initial 11-Torr increase in Paco 2. A second exposure 2 h later resulted in simple hypoventilation and increased Paco 2 only 4.3 Torr. Carotid body hypocapnia had no effect on the ventilatory sensitivity to Paco ~ in 5 of the 7 Group 3 goats. In these animals, lowering the Pcbco~. 11 Torr did not significantly change the slope of the systemic CO2 response for VE, VT and f, but significantly increased the Paco2-intercept values for "¢E and VT (table 2). Thus, the non-CB chemoreceptor response to COz was not blunted by CB hypocapnia, but was shifted to the right. During periods of unstable breathing in CB hypocapnia, the effective ventilatory sensitivity to systemic CO: was greatly diminished. In 2 of the 7 Group 3 goats studied, Pcbco 2 : 3 7 . 6
Torr
Pcb¢o 2
31.8
Torr
Pa¢o 2 = 6 8 . 1 T o r r
Paco 2 = 4 1 . 9 T o r r VT 0.5L
[
30 s
Fig. 3. Ventilatory record from one goat during CB normocapnia and during CB hypocapnia with increased FIc¢ 2. Breathing remained unstable in CB hypocapnia when Paco 2 was elevated.
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L. DARISTOTLE et al.
CB hypocapnia could produce apneas or irregular ventilatory patterns, and this instability persisted as P a c o 2 was increased (fig. 3). These measurements of breathing were therefore not well-correlated (r 2 < 0.70) with P a c o 2 during CB hypocapnia. In one of these 2 goats, a second exposure to CB hypocapnia later in the experiment resulted in a stable hypoventilation and a brisk P a c o 2 response.
Discussion Perfusion of the vascularly isolated CB with hypocapnic blood resulted in significant hypoventilation in awake and anesthetized goats, an effect which was abolished by CB denervation in anesthetized goats. This hypoventilation was associated with a CB hypocapnia-induced shift of the systemic (central chemoreceptor) response toward higher Paco2S in most awake goats studied, and with ventilatory instability in some awake goats. Critique of methods. Separation of CB and brain blood flows is a characteristic of our CB perfusion model which was essential in these studies. We are confident that central neural structures were not exposed to the blood gas conditions delivered to the CB. Validation studies have been reported previously (Busch et al., 1985) in which no evidence of significant mixing between blood perfusing the CB and that perfusing the brain was detected. In addition, we injected radiopaque dye into the CB perfusion cannula in one of the animals used in these experiments and radiographic angiography confirmed that the CB circulation did not include brain vasculature. The aortic bodies were intact in our preparation and were exposed to the systemic circulation; thus, they potentially could have contributed to the ventilatory responses to elevated P a c o 2 in these experiments. We minimized aortic chemoreceptor effects during the CO z responses by maintaining systemic hyperoxia. We also tested for extra-CB peripheral chemosensitivity by intravenous administration of NaCN (50 #g/kg) that was restricted from the CB circulation by the perfusion circuit; no hyperventilatory response to NaCN was found in these animals, indicating absence of significant extra-CB peripheral chemoreflexes. Thus, we feel that aortic chemoreceptor effects are not important in our results. We used simple linear regression to estimate the slopes of the ventilatory responses to CO2 in this study. Although VT and f may not be truly linear functions of P a c o 2, the mean coefficients of determination (r 2) for VE, VT and f were greater than 0.70 in 5 of 7 Group 3 goats. We conclude that these simple linear models were an adequate fit for our data and provided useful information about these responses. CB hypocapnia and hypoventilation. Carotid body hypocapnia produced a hypoventilatory response in goats similar to that observed by Heymans et al. (1930) and Fitzgerald et aL (1964) during perfusion of carotid chemoreceptors with hypocapnic blood in anesthetized dogs. Our unanesthetized goats exhibited a 24~o depression of VE from
VENTILATION IN CAROTID BODY HYPOCAPNIA
131
control during CB hypocapnia, comparable to the 24~o decrease seen in the dogs of Fitzgerald et al. (1964). The major effect of CB hypocapnia on ventilation in goats was a reduction of tidal volume. This is compatible with evidence that the ventilatory response to changes in Paco 2 is mostly due to changes in tidal volume or phrenic amplitude (Davis and Stagg, 1975; Speck and Webber, 1985). In addition, data on awake hypercapnic men exposed suddenly to transient hypocapnia revealed that the resulting peripheral chemoreceptormediated decrease in ventilation was produced more by decreases in tidal volume than by decreases in respiratory rate (Miller et al., 1974). The hypoventilation demonstrated during selective perfusion of the CB with hypocapnic blood was most likely a carotid chemoreceptor effect. Carotid chemodenervation abolished the ventilatory consequences of CB hypocapnia in our anesthetized goats and in anesthetized dogs (Fitzgerald et al., 1964). In addition, single chemoreceptor unit discharge frequency recordings during CO 2 response curves obtained in our laboratory from anesthetized goats suggest that ventilatory drive from CB chemoreceptor input is very low at the levels of Pcbco: achieved in this study (Engwall et al., 1988). In 12 single fibers, plots of discharge frequency vs. Paco2 show a mean Paco2-intercept of 25.1 Tort at 0 imp/sec, approximating the Pco: values producing ventilatory depression in CB-perfused goats. Although actual discharge rates did not reach zero, the mean discharge rate of 5 fibers studied in the hypocapnic range was 15~ of the mean normocapnic discharge rate (0.22 imp/sec at Paco2 = 20.1-28.0 Ton" vs. 1.48 imp/sec at Paco ~ = 40 Torr). Carotid sinus nerve activity was also found to be much reduced or eliminated by systemic hypocapnia in anesthetized cats (Samaan and Stella, 1935). It is interesting to note that CB denervation resulted in a less marked depression of ventilation than did CB hypocapnia (table 1). One might expect that removal of CB chemoreceptor input by denervation would produce at least equivalent ventilatory effects as the decreased CB chemoreceptor afferent traffic associated with CB hypocapnia. One possible explanation for this disparity would be the ventilatory effects of elimination of baroreceptor input in sectioning the carotid sinus nerve. Ventilation has been shown to increase with decreasing carotid sinus baroreceptor stimulation via decreasing isolated sinus pressure in anesthetized dogs (Brunner et al., 1982). Interruption of carotid sinus baroreceptor afferents in our CB denervations might likewise stimulate breathing and counteract the depressant effect of loss of CB chemoreceptor drive. Perfusion of the neurally intact CB, however, should have little effect on baroreceptor function, and the baroreceptor contribution to the ventilatory response to CB hypocapnia should be negligible. The degree of hypoventilation observed during CB hypocapnia varied considerably among goats. This response was not dependent upon the magnitude of change of PcbcQ (fig. 4); the animals exhibiting the largest decreases in ventilation were not those subjected to the largest falls in Pcbco ~. The rate of change and the 'shape' of the decline in Pco~ at the CB chemoreceptor have been shown to affect ventilation (Dutton et al., 1967), but we cannot address the importance of these factors as we lack this information for our model.
132
L. DARISTOTLE et al. 0 • •
-25
•
co
-50 -75 -100 ~ o -20
t
i
-15
-10
A P°.o.,
i
-5 (Torr)
0
Fig. 4. Decreases in ventilation as percentages of control values produced by decreases in Pcbco 2in awake goats, Groups 1 and 3 (.) and anesthetized goats, Group 2 (o). Ventilatory depression did not appear to be related to the magnitude of CB hypocapnia in these animals. Note that 3 of the 5 anesthetized goats became apneic ( - 100~o A~'E)when APcbco2 = - 15 to -20 Torr. Peripheral chemosensitivity t o C O 2 may partially account for the trends seen in our data. Individual ventilatory responses to CB hypercapnia performed in 5 goats from Group 1 correlated well with responses to CB hypocapnia ( r 2 > 0.80); goats with greater depression of ventilation in CB hypocapnia tended to hyperventilate more in CB hypercapnia. This relationship suggests that ventilation may be a linear function of P c b c o 2 in the range examined in some goats. Central chemoreceptor responses will also determine the level of ventilation during CB hypocapnia. The dependence of the respiratory controller on peripheral vs. central chemoreceptor CO2 sensitivities may be important here, and may vary widely between animals (Heeringa et aL, 1979). Our data suggests that CB hypocapnia did not interact with central chemosensitivity in producing hypoventilation. In the majority of goats studied, CB hypocapnia had no significant effect on the slope of the extra-CB CO2 ventilatory response, but resulted in a shift of the response toward lower ventilatory and higher P a c o 2 values. We have previously shown in this same model that the ventilatory response to hypercapnic-hypoxic CB perfusion, like that from hypocapnic perfusion, is integrated with central chemosensitivity in an additive manner (Daristotle and Bisgard, 1987). The greater degree of ventilatory depression seen in anesthetized goats may have been, in part, the result of chloralose increasing the Pco2 at which apnea occurred (Florez and Borison, 1969). In this manner, CB hypocapnia of similar magnitude could elicit simple hypoventilation in an awake animal and apnea in a chloralose-anesthetized one. CB hypocapnia and ventilatory instability. Carotid body hypocapnia produced large oscillations in ventilatory timing and volume in a minority of Group 1 and 3 goats studied. Many factors have been proposed to affect respiratory stability, including
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chemosensitivity, conscious state, body gas stores, and circulatory effects (Cherniack and Longobardo, 1986). We suggest that the highly variable breathing patterns seen in CB hypocapnia could be due to a combination of systemic hypercapnia, central chemosensitivity, CB chemosensitivity and spontaneous ventilatory oscillations. Hypercapnia can contribute to ventilatory instability by increasing the effectiveness of ventilation in changing arterial blood gas tensions. When resting Paco 2 is elevated, a change in ventilation will produce a greater change in Paco 2because of the hyperbolic shape of the metabolic curve. This increase in the gain of the control of Paco 2 would increase the gain of the overall negative feedback control loop relating ventilation and Paco 2, and could result in system instability and oscillations in ventilation (Chapman et al., 1988). Carotid body hypocapnia decreased ventilation and increased Paco 2in our goats, and therefore could predispose to such oscillations. Indeed, the 4 goats with irregular breathing in CB hypocapnia also showed the greatest initial changes in Paco 2. In addition, ventilatory instability was associated with a larger increase in Paco ~ than was stable hypoventilation when one goat was subjected to CB hypocapnia twice. This systemic hypercapnia would be sensed chiefly through the central medullary chemoreceptor; the CB chemoreceptor was exposed only to steady-state hypocapnia. Central chemosensitivity would therefore dictate the response to the increase in Paco ~. An enhanced systemic CO 2 response could lead to respiratory oscillations by increasing the feedback loop gain. Although our data are insufficient to fully evaluate the role of central chemosensitivity in CB hypocapnic ventilatory instability, the (CB normocapnic-normoxic) systemic CO2 ventilatory response slope of one of the 4 goats was greater than the mean slope (2.79 vs. 2.29 L/min/Torr), and could have contributed to the increased variability of breathing during CB hypocapnia in this animal. Carotid body chemoreceptor function could promote unstable breathing in CB hypocapnia. In our experiments, the CB was exposed to steady-state normoxic hypocapnia, and could not respond to changes in blood gas tensions resulting from alterations of ventilation. Because stability of respiratory output is thought to be dependent on the controller's ability to be informed of the consequences of its actions (Cherniack and Longobardo, 1986), this effective lack of CB sensitivity to the chemical sequelae of ventilatory changes, combined with the reduction in tonic CB chemoreceptor input to the controller, may result in ventilatory instability during CB hypocapnia. We observed spontaneous cycles in the pattern of breathing during CB normocapnicnormoxia in one of the goats that developed ventilatory instability in CB hypocapnia. Such spontaneous oscillations have been observed more frequently in sleeping humans which developed periodic breathing in hypoxia than in those which did not (Chapman et al., 1988). These oscillations could be magnified when feedback loop gain is increased (by systemic hypercapnia in our goats or hypoxia in the sleeping humans), and then lead to marked ventilatory instability. It is possible that changes in the state of arousal could be involved in the irregular breathing observed in CB hypocapnia. Decreases in the level of consciousness might facilitate apnea and instability, and variation in alertness could explain, at least in part, the variation between goats and within experiments in the ventilatory effects of CB
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h y p o c a p n i a . H o w e v e r , w e did n o t e v a l u a t e c o n s c i o u s state in o u r g o a t s in t h e s e studies, a n d t h e r e f o r e c a n n o t s u p p o r t o r refute this theory. W e d o n o t feel it is likely that t r a n s i e n t c h a n g e s in C B p e r f u s a t e c o n d i t i o n s c o u l d be the s o u r c e o f the v e n t i l a t o r y instability. A l t h o u g h w e did n o t m a k e c o n t i n u o u s m e a s u r e m e n t o f C B b l o o d p H a n d gas t e n s i o n s , t h o s e v a l u e s w e o b t a i n e d w e r e stable during the h y p o c a p n i c trials, a n d c h a n g e s in v e n t i l a t i o n did n o t t r a c k small c h a n g e s in C B conditions.
Acknowledgements.The authors wish to thank Mr Gordon Johnson, Ms Margaret Rankin and Ms Kristine Bisgard for their valuable technical assistance. This work was supported by USPHS grants HL15473, HL07052 and HL07655.
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