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J Appl Physiol 87: 1026-1034, 1999;
8750-7587/99 $5.00
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Vol. 87, Issue 3, 1026-1034, September 1999

Effect of carotid body denervation on breathing in neonatal goats

T. F. Lowry, H. V. Forster, L. G. Pan, M. A. Korducki, J. Probst, R. A. Franciosi, and M. Forster

Departments of Physiology and Pediatrics, Medical College of Wisconsin, Zablocki Veterans Affairs Medical Center, and Program in Physical Therapy, Marquette University, Milwaukee, Wisconsin 53226


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The objective of the present study was to determine in goats whether carotid body denervation (CBD) at 1-3 days of age causes permanent changes in breathing greater than those that occur after CBD in adult goats. Goats underwent CBD (n = 6) or sham CBD (n = 3) surgery at 1-3 days of age. In addition, one unoperated control animal was studied. Bolus intravenous injections of NaCN 2 days postsurgery verified successful CBD surgery. However, at 3, 11, and 18 mo of age, the CBD goats had regained a NaCN response that did not differ (P > 0.10) from that of intact goats. Intracarotid NaCN injections elicited a hyperpnea in the sham CBD but not the CBD goats. Only one animal exhibited highly irregular breathing [characterized by prolonged (>9-s) apneas] after CBD, and the irregularity disappeared by 3 mo of age. One CBD goat died at 35 days of age, and autopsy revealed that death was associated with pneumonia. After 3 mo of age, there were no statistically significant differences (P > 0.10) between sham and CBD goats in eupneic breathing, hypoxia and CO2 sensitivity, and the exercise hyperpnea. It is, therefore, concluded that CBD at 1-3 days of age in goats does not appear to affect selected aspects of respiratory control after 3 mo of age, conceivably because of the emergence of other functional chemoreceptors that compensate for the loss of the carotid chemoreceptor.

carotid chemoreceptors; breathing; neonates


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INVESTIGATORS HAVE REMOVED the peripheral chemoreceptors (carotid or both carotid and aortic bodies) in several species of mammals before or shortly after their birth to determine the contribution of these receptors to 1) control of breathing and 2) the development of normal adult respiratory control (4, 5, 7, 10, 14-17). The results of studies in which carotid body denervation (CBD) was performed in utero showed that the chemoreceptors are not necessary for fetal breathing movements or the initiation of breathing after birth (14-17). The results of studies in which CBD was performed postnatally confirm the hypothesis that the carotid chemoreceptors contribute to ventilatory control in the immediate postnatal period (4, 5, 7, 10). Denervated neonates breathe more irregularly (5, 7, 10) and hypoventilate (4, 7, 10). In addition, a high degree of mortality was observed in neonatal CBD (nCBD) studies (4, 7, 10). It was suspected that death was due to ventilatory control abnormalities, which led Donnelly and Haddad (7) and Haddad et al. (9) to hypothesize that carotid chemoreceptor activity was necessary for the normal development of medullary respiratory control. This hypothesis was suggested by studies on the effect of eye closure on the development of the visual cortex (25). Specifically, in the visual system some neural connections develop in the postnatal period, whereas others are established before birth (11, 12, 25). The postnatal maturation of these connections is dependent on neural activity during a critical postnatal period within the developing system. In addition, the effects of neural silencing (eye closure) have been shown to extend through several synapses, implying that silence (dysfunction) in one neural path could have a widespread effect on a developing system (25). It is not known if these observations can be generalized to respiratory control in early postnatal life.

Cote et al. (5) noted a "window of vulnerability" in relation to the age at which CBD produced significant breathing irregularities. These findings have implications for the study of sudden infant death syndrome, because the incidence of sudden infant death syndrome peaks at an age of 3-4 mo (8). The nCBD studies add to the body of literature suggesting that respiratory control is immature at birth. However, only one of the previously cited studies (4) followed the impact of denervation for more than a few weeks after denervation. Bureau et al. (4) concluded that denervation did produce an effect on maturation, but it was unclear whether the effect was because of "blocked maturation" or was simply a denervation effect, because no reference was made to the acute effect of CBD in adult sheep.

The central focus of the present study was to determine in goats whether CBD at 1-3 days of age causes permanent changes in breathing greater than that which occurs after CBD in adult goats. In other words, because attenuation of visual afferents through eye closure in neonates causes long-lasting visual defects, will CBD (and presumed attenuation of carotid chemoreceptor afferents) cause long-lasting changes in breathing?


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Before initiation of any studies, all protocols and procedures were reviewed and approved by the Medical College of Wisconsin Animal Care Committee.

Animals

We obtained seven pregnant does, which were allowed to deliver naturally in our animal care facility. Six neonatal goats were studied as CBD, three as sham-operated controls, and one as an unoperated control. Neonates were operated on within the first 4 days of postnatal life. Neonates were kept with the does and allowed to nurse ad libitum until 3 mo of age.

Surgery

Anesthesia was induced with ketamine (15 mg/kg im), and the goats were intubated. Anesthesia was maintained with halothane (1-2%) in 100% O2, sufficient to prevent arousal in response to surgical manipulations. Surgery was performed as previously described (19). Briefly, a single midline incision was made, and the muscles adjacent to the larynx were retracted to expose the carotid sinus region. In goats, the carotid bodies lie on the interior wall of the occipital artery. Ties were placed on the occipital artery adjacent to the common carotid and ~1 cm distal to the bifurcation, and the portion of the artery between the ties was excised. This procedure was then repeated on the second side. In sham surgeries, the carotid sinus region was exposed, but no ties were placed.

Experimental Design

Several earlier nCBD studies had reported a high rate of mortality (4, 7, 10). In addition, there was evidence that the system under study was immature at birth, developed during the study period, and was subject to postnatal environmental influences (3, 11, 14, 21, 25). Furthermore, studies of the development of the visual cortex suggested that "environmentally induced" alterations of normal development were essentially permanent (11, 25). Because of these considerations, we studied nCBD animals minimally in the immediate postsurgical period. The intent was to minimize stress other than CBD, thereby permitting us to determine whether CBD in the neonatal period permanently changes the control of breathing. We assumed that effects such as those observed in the visual cortex would still be evident months to years post-nCBD.

Assessment of Completeness of Denervation

One to two days postsurgery, all animals received intravenous (jugular vein) bolus injections of 0.1 mg/kg NaCN to assess completeness of denervation. The ratio of ventilation in the period 10-30 s after injection to control ventilation was determined for each injection. The average response to several injections was determined for each animal. The intravenous response to NaCN was reassessed at the ages of 3, 11, and 18 mo. At the same ages, unilateral (common carotid) arterial injections of 0.01 mg/kg NaCN were given to assess whether peripheral chemoreception had redeveloped at the carotid bifurcation. The ratio of ventilation postinjection (5 breaths after injection) to control (5 breaths preceding injection) was assessed for each trial, and an average was obtained for each animal.

Assessment of Respiratory Regularity

After surgery, animals were placed in a whole body plethysmograph to monitor ventilation while breathing room air. This monitoring was confined to periods of <1 h and occurred once per week during the first month after surgery. These data allowed a gross assessment of breathing regularity. If apneas were commonly observed (>1 in 10 min), and the length of apnea exceeded 10 s, the animal was considered irregular. Apneas after sighs were not considered signs of irregularity. Observation sessions usually included periods of quiet wakefulness and one or more stages of sleep. Periods of rapid-eye-movement (REM) sleep (eyes closed and twitching, and jerking body movements) were sometimes observed but did not comprise a large proportion of the observation time.

Carotid Elevation Surgery

When the animals were 3 mo of age, surgery was performed to elevate a carotid artery so that arterial blood gases and pressure could be monitored. The kids were trained to accept a muzzle mask so that hypoxic and hypercapnic responses could be accurately characterized, and they were trained to walk on a treadmill. The responses to hypoxia, hypercapnia, and exercise were assessed at 3, 11, and 18 mo of age.

Ventilatory Responses to Hypoxia and Hypercapnia

Ventilation was monitored with a muzzle mask/breathing valve combination, with a pneumotachometer (Hans Rudolph) attached to the inspired side of the breathing valve. The signal from the pneumotachometer was transduced with a Validyne pressure transducer/demodulator, fed into a computerized data-collection system (Codas), and integrated to yield volume per unit time. Blood pressure was monitored, and blood samples were taken from a chronic catheter placed in an elevated carotid artery. The blood pressure signal was monitored with Statham P23 transducers. A Grass polygraph (model 7d) was used to condition both ventilation and blood pressure signals before they were recorded on Codas. Blood-gas samples were analyzed on a Corning blood-gas analyzer (model 278). Blood-gas analysis was repeated until two readings of arterial PCO2 (PaCO2) agreed to within 0.6 Torr.

Control data were obtained while the (awake) goats quietly stood or lay in a stanchion, breathing room air. Ventilation was monitored for at least 5 min. Two blood-gas samples were drawn over the final 2 min of the control period. Arterial blood pressure was monitored continuously except for the time during which blood-gas samples were taken.

In the hypoxia protocol, the inspired gas was changed to a hypoxic mixture for 10 min. Blood-gas samples were obtained during the final 2 min of hypoxia. At 3 mo of age, the inspired O2 fraction (FIO2) of the hypoxic mixture was 0.10. At 11 and 18 mo of age, the FIO2 of the hypoxic mixture was 0.12.

In the hypercapnia protocol, the inspired gas was elevated stepwise to inspired CO2 fractions of 0.03, 0.05, and 0.07 (FIO2= 0.21, balance N2) for 5-min periods each. Blood-gas samples were obtained over the final 2 min at each level of inspired CO2 fraction. Breathing and blood pressure were monitored for an additional 5 min after the inspired gas was returned to room air.

Ventilatory Response to Exercise

Two different exercise protocols were utilized. In the first, goats were fitted with the muzzle mask/breathing valve apparatus so that ventilation and metabolic rate could be monitored. Mixed expired gases were collected in a Tissot spirometer and analyzed with calibrated O2 and CO2 gas analyzers (models S-3A/I and CD-3A, respectively, Applied Electrochemistry). Ventilation was calculated from the Tissot displacement readings taken at minute intervals. After a 5-min control period, the treadmill was started at 1.8 miles/h, 5% grade. After 5 min at this workload, the treadmill grade was increased to 15% for another 5 min. Metabolic rate was determined from expired gas collections made at the end of control and over the last 30-60 s of each workload.

In the second exercise protocol, we determined the temporal pattern of PaCO2 during the same exercise regimen. This protocol was adopted because it allows a more detailed assessment of how well subjects match ventilation to metabolic rate at the onset of and during workload transitions as well as steady states. In addition, goats were not encumbered with the mask apparatus during this protocol. Four control blood samples were drawn during a 2-min control period. At the start of exercise, blood was drawn into a waste syringe to flush the catheter for 15 s, and then three 15-s blood samples were drawn. During the next 2 min, four more samples were drawn over periods of 30 s each. One sample was drawn during the final minute at each workload. At the end of the first workload, the treadmill grade was increased, and the blood sampling protocol was repeated.

Data Analysis and Statistics

All experiments were performed in duplicate, and a mean was obtained for each animal. Means reported for each denervation group were obtained by averaging the values from each animal. SPSSPC was used to perform statistical analysis on the data.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Assessment of Denervation Status

The response to intravenous NaCN injections was significantly (P < 0.05, t-test) lower in the carotid denervated group than in the intact group 1-2 days postsurgery (Table 1). However, at 3, 11, and 18 mo of age, the responses of intact and denervated goats no longer differed (P > 0.10). The responses at these three ages were greater than those determined immediately after surgery and were essentially at adult levels. This trend was evident in both denervated and nondenervated groups. The response to common carotid injections of NaCN was substantial in the intact group, but there was virtually no response in the CBD group (Table 1).

                              
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  		Table 1.   Arterial and venous NaCN ventilatory response ratios in goats

Mortality

Two neonates involved in our study died. One was a sham-operated control animal that was the "runt" of a set of triplets. This animal did not nurse well and died, apparently of nutritionally related causes. The other mortality was a CBD animal that appeared normal in the first month after surgery. Weight gain was normal, the intravenous NaCN response was not among the lowest, and behavior appeared normal. Thirty-five days postsurgery, the animal was observed to be listless. The animal suffered an apparent seizure but then appeared to recover. Respiration appeared regular and was not labored. Rectal temperature was not elevated. The next morning, the kid was found dead in its cage. An autopsy revealed no pathology of the heart or brain, but examination of the lungs revealed the animal had pneumonia.

Resting Blood Gases, MAP, and HR

Resting values for arterial pH, blood gases, mean arterial pressure (MAP), and heart rate (HR) were obtained while the animals stood quietly on a treadmill (Table 2). There were no significant changes in PaCO2 or pH with age in intact or CBD animals, and there was no significant difference between intact and CBD animals (P > 0.05). MAP and HR were unaffected by CBD, except that MAP was significantly lower in the CBD group at 11 mo of age (P < 0.05). HR declined significantly (P < 0.05) from the value at 3 mo but did not continue to decline (P > 0.05) from 11 to 18 mo of age.

                              
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  		Table 2.   Physiological variables of carotid intact and denervated (CBD) goats at 3, 11, and 18 mo of age

Pattern of Breathing

Neonatal goats were monitored in a whole body plethysmograph at least once a week for 1 mo after surgery. Neonates were studied while awake (eyes open), asleep (eyes closed, no eye or body movements), and during apparent REM sleep (eyes closed and twitching). Breathing typically decreased at the transition from awake to asleep and became more irregular during apparent REM episodes. Visual inspection of the data showed that apneas were not a regular feature of the breathing pattern of intact neonates. One CBD neonate did exhibit a high degree of respiratory irregularity in the month after surgery (Fig. 1). The irregularity was not confined to sleep and was not observed on all days. However, the other five CBD neonates, including the neonate that died 35 days postsurgery, did not exhibit such irregularities. At 3, 11, and 18 mo of age, there were no significant (P > 0.10) differences in pulmonary ventilation, tidal volume, breathing frequency, and inspiratory and expiratory time between CBD and adult goats breathing room air at rest (Tables 2 and 3). Moreover, there were no differences (P > 0.10) between the two groups in the coefficient of variation of tidal volume and inspiratory and expiratory time (Table 3).


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  		Fig. 1.   Respiration tracing from carotid body denervation (CBD) goat in whole body plethysmograph. Inspiration is represented by upward deflections. Goat was denervated at 4 days of age, and tracing is from 34 days of age. Highly irregular breathing seen here was no longer evident when goat was restudied at 3 mo of age.


                              
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  		Table 3.   Selected breathing variables in a subset of individual carotid intact and denervated (CBD) goats at 3 mo of age

Ventilatory Responses to Hypoxia

Intact and CBD goats of all ages exhibited similar responses to hypoxia. All ages of intact and CBD goats increased ventilation during the first 2-3 min of hypoxia and then maintained a relatively constant, elevated ventilation, which did not differ (P > 0.05) between the two groups (Fig. 2). The decrease in PaCO2 (rest to minutes 7-9 of hypoxia) did not differ between intact and CBD groups at any age (P > 0.05) (Table 4).


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  		Fig. 2.   Ventilatory response to hypoxia (means ± SE) in intact () and CBD (black-triangle) goats at 3 (A), 12 (B), and 18 mo of age (C). Ventilation (VE) is normalized to control (VE/control VE) to facilitate comparison of the different ages. Level of hypoxia at 3 mo of age was inspired O2 fraction (FIO2) = 0.10, and at 11 and 18 mo of age FIO2 = 0.12. Duration of hypoxia was 10 min followed by 5 min of normoxia.


                              
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  		Table 4.   Control and hypoxic blood-gas measurements at ages 3, 11, and 18 mo

Ventilatory Responses to Hypercapnia

There were no significant (P > 0.10) differences between intact and CBD animals in CO2 sensitivity (ratio of change in inspired ventilation to change in PaCO2), and there were no significant changes with age (Fig. 3). The observed values are within the previously reported normal range in adult goats (19).


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  		Fig. 3.   Hypercapnia response slopes (l · min-1 · kg-1 · Torr PaCO2-1, where PaCO2 is arterial PCO2) of intact (striped bars) and CBD (open bars) goats at 3, 11, and 18 mo of age. Slopes are not different among age or denervation groups and fall within normal range of variation of intact adult goats.

Ventilatory Responses to Exercise

Ventilatory responses to exercise did not differ (P > 0.10) between intact and CBD groups (Figs. 4 and 5). At 11 mo of age, the exercise protocol showed that CBD did not alter the ventilation/metabolic rate relationship (Fig. 4). Intact animals exhibited a modest hyperventilation at the onset of exercise, whereas a slightly exaggerated hyperventilation was seen in CBD animals (Fig. 5).


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  		Fig. 4.   Pulmonary VE, tidal volume (VT), and breathing frequency (f) are plotted against metabolic rate [O2 consumption (VO2)] for exercise at 11 mo of age. open circle , CBD; , intact goats. There were no significant (P > 0.10) differences between the two groups in these exercise responses.



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  		Fig. 5.   PaCO2 during exercise in CBD (open circle ) and intact () goats at 11 mo of age. Although CBD goats exhibit higher (but not significantly) resting PaCO2, they appear to hyperventilate to same level of PaCO2 at onset of and during exercise. Elevated resting PaCO2 and an accentuated response to exercise has been previously reported in CBD studies (2, 18-20). MPH, miles/h.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study show that, in goats that had CBD or sham CBD at 1-3 days of age, there were minimal differences in breathing between the sham and CBD goats at 3, 11, and 18 mo of age; therefore, CBD in this neonatal period does not cause permanent changes in control of breathing greater than those observed after CBD in adult mammals.

Limitations

The focus of this study was on the effect on breathing, after 3 mo of age, of CBD in goats <4 days old. As already stated, extensive studies on animals <3 mo of age were avoided, because these studies may potentially affect development and/or recovery from surgery. In other words, we compromised between maintaining near normal physiological conditions and detecting differences between intact and CBD goats over the first 3 mo of life. We chose goats for this study primarily because our laboratory has previously documented the acute and chronic effects on breathing of CBD in adult goats (19). We have studied the effects of CBD at only one age primarily because of the limited availability of newborn goats and our own limited capability for maintaining a large number of chronic CBD goats. We limited the scope of the studies, resulting in several unanswered questions (such as site of residual chemosensitivity), because we felt that answering such questions required a different, specific focus. As a result of these limitations, the conclusions of the study are specific to goats carotid denervated at one age and the status of breathing after 3 mo of age.

Plasticity of arterial chemoreception. Partial return of peripheral chemosensitivity after CBD has been reported in a number of species (1, 19, 23). Such an effect has been termed "plasticity," which refers to situations in which neurally mediated behavior is lost because of nerve section but regained over time after surgery. When a nerve has been cut, information from distal sense organs can no longer reach the site(s) where the information is processed or relayed, and reflex actions that arise from stimulation of the sense organs no longer occur when the sense organs are activated. If reflex responses to a stimulus are restored, it can be inferred that either the sense organ has been reinnervated (or remaining innervation has been strengthened) or that the response has been "relearned," i.e., the response is now mediated by another set of receptors. Potential mechanisms responsible for plasticity, in the present context, range from de novo synaptic formation to synaptic strengthening (sensitization). The "return of chemosensitivity" may simply be the result of stronger synaptic connections (either numerically or on an individual basis) between the remaining (residual) chemoreceptors and the central neurons that mediate the response.

The carotid chemoreceptors are generally regarded as being the primary sensory organ responsible for the acute reflex increase in ventilation that occurs in response to hypoxia or to venous NaCN injection. However, others have noted that, when the carotids are removed in adult animals, a significant amount of hypoxic and NaCN responsiveness returns over time (1, 2, 19, 23). A study of adult CBD cats found a significant restoration of the hypoxic response 30-43 days after denervation, and by 260-315 days the hypoxic response was essentially normal (23). Adult carotid and presumably aortic-denervated ponies recovered 30-40% of the normal response to acute hypoxia and NaCN by 22 mo postsurgery (1). Studies conducted after subjects had regained substantial peripheral chemosensitivity showed that these responses did not originate from the carotid region but that sectioning the vagosympathetic trunk abolished the regained response in the cat and sectioning the aortic nerve substantially reduced the response to intravenous NaCN in the ponies. The conclusion from these studies was that the regained chemosensitivity was from a site other than the carotid body site.

In the present study, we also found that the response to NaCN was nearly eliminated 1 or 2 days after CBD, but, by 3 mo of age, the response to venous NaCN injections did not differ between CBD and sham CBD goats. However, only the sham CBD goats at 3, 11, and 18 mo of age had a response to intracarotid injection of NaCN. Nevertheless, it is conceivable that regeneration of chemosensitivity in the carotid region could account for the regained response to intravenous NaCN if the blood supply to the regenerated area is not through the carotid arteries. The data summarized above (1, 23) do not support this possibility; thus we believe it likely that the regained response in these CBD goats is not mediated by carotid chemoreceptors.

Cote et al. (5) reported that CBD in neonatal piglets (4-22 days of age) eliminated the normal hypoxic hyperventilation. However, the responses were measured after a gradual (15- to 20-min) induction of hypoxia, which would decrease the probability of finding an increase in ventilation and decrease in PaCO2 because of the well-known phenomenon of hypoxic ventilatory roll-off. In addition, the hypoxia protocols were carried out only 1-2 wk postdenervation and on the day after instrumentation surgery.

In response to hypoxia, adult intact goats slightly increase ventilation, resulting in a small decrease in PaCO2 (19). Two weeks after CBD in adult goats, there is an even smaller increase in ventilation and decrease in PaCO2. It is important to note that hypoxic responsiveness and arterial chemosensitivity are not entirely eliminated as a result of CBD in adult goats. As in the neonatal goats, the residual chemosensitivity in adult goats does not apparently originate in the carotid region. Accordingly, neonatal and adult goats, adult cats and ponies, and probably piglets demonstrated plasticity in arterial chemosensitivity after CBD.

Mortality and breathing irregularity. A neonate died in both the intact and CBD study groups. Neither appeared to exhibit the highly irregular breathing reported by others, and probable causes of death were unrelated to respiratory control. In previous nCBD studies, Donnelly and Haddad (7) reported five of nine CBD piglets had died, and Bureau et al. (4) reported that three of seven CBD lambs died several weeks post-CBD. Hofer (10) performed both carotid and aortic denervation in neonatal rats and found that 11 of 24 died within 3-18 days after surgery. Donnelly and Haddad (7) also denervated both carotid and aortic chemoreceptors in other neonatal piglets and found the mortality was not greater than with only carotid denervation. Of these investigators, only Bureau et al. (4) performed autopsies on the animals that died. They reported "focal atelectasis with a minor degree of inflammation," which is consistent with aspiration pneumonia and could have contributed to the deaths.

The rate of mortality (1 of 6) in the present study is less, although not significantly so, than the rates reported by others. It is important to note that the observed mortality is not associated with "respiratory pump control" but apparently with aspiration pneumonia, implying upper-airway dysfunction. It is also worth noting that Cote et al. (5), who did not observe any fatalities, remarked on the importance of intubating the piglets during surgery and for up to 1 h postsurgery to avoid "asphyxia secondary to prolonged apnea," especially in the animals denervated at 9-10 and 12-15 days of age. This observation implies upper airway dysfunction, not central apnea. These age groups are the same as those that Cote et al. reported to exhibit prolonged apneas in studies done 1-2 wk postsurgery.

Neonatal goats were observed carefully to determine whether apneas were a regular feature of their breathing, as had been reported in several other nCBD studies (4, 5, 7, 10). Animals frequently slept for part of the sessions; hence the observations can be taken as applicable to all arousal states. Although one CBD goat did exhibit some prolonged apneas, the irregularity disappeared and was not noted in later sessions. Moreover, pattern and variability in breathing did not differ between sham and CBD goats after 3 mo of age (Tables 2 and 3). These data are sufficient to establish that breathing irregularity of the type and frequency observed by others was not present in this study.

We cannot exclude the possibility that the reduced levels of mortality and irregular breathing are due to "species differences," e.g., the goat may be more mature at birth and/or better able to tolerate the experimental protocol. In addition, the lack of significant effects in this study may be related to the age at which the denervations were performed. Cote et al. (5) did not report significant irregularities in piglets denervated at 4-5 days of age but found profound irregularities when denervations were performed at 12-15 days of age. Because their piglets were only studied at one time point in the postdenervation period, it is not known whether such effects were permanent.

Even though we found relatively low mortality and minimal irregular breathing among CBD goats, the single fatality again raises the issue of whether attenuated carotid chemoreception might be a factor contributing to sudden infant death. Our data suggest that, if this attenuation is a factor, it is not necessarily related to abnormal ventilatory control leading to irregular breathing, as suggested by others (4, 7), because there was no irregular breathing in the goat that died.

Resting blood gases. Eupneic PaCO2 was not significantly elevated in nCBD goats 3, 11, and 18 mo after surgery, indicating that they were not hypoventilating. Data from previous studies vary as to whether "age at time of denervation" affects the magnitude of hypoventilation. Donnelly and Haddad (7) found a 9- to 12-Torr increase in PaCO2 1 day postsurgery when either the carotid or both carotid and aortic chemoreceptors were denervated at 3-9 or 30 days of age. Cote et al. (5) found no increase in PaCO2 1-2 wk postsurgery when animals were denervated at 4-5 days of age but found significant increases at 9-10, 12-15, and 21-22 days of age. The largest increase in PaCO2 (~10 Torr) was noted in the oldest age group. Cote et al. speculated that a fetal respiratory control mechanism persisted into early postnatal life and remained functional if the carotid chemoreceptors were removed shortly after birth. It is possible that goats denervated at older ages would similarly exhibit a greater hypercapnia and greater respiratory disturbances.

We found in adult goats that eupneic PaCO2 increased after CBD, reaching a maximum hypercapnia of 11 Torr above control 4 days after CBD (19). PaCO2 then returned toward control, remaining slightly elevated (3.5 Torr) 2 wk later. Previously, it had been observed that eupneic PaCO2 was elevated after CBD (1, 2, 18, 20) and after carotid and aortic denervation remained elevated for 22 mo (1). The prolonged hypercapnia in the latter study may indicate that, after CBD alone, aortic chemoreceptors may contribute to eventual normalization of PaCO2. More rapid compensation in the neonate may reflect either persistence of fetal mechanisms or the well-known enhanced plasticity of young animals.

Ventilatory responses to hypercapnia and exercise. No significant differences in the response to elevated inspired CO2 were noted between nCBD and intact goats, at any age studied. In addition, there was no age-related change in CO2 sensitivity during the study period in either intact or nCBD goats. However, CO2 sensitivity may have decreased and returned to normal before our first post-CBD studies.

In a study of the acute influence of CBD on ventilatory control in adult goats (19), a transient decrease in sensitivity to inspired CO2 was noted, which temporally corresponded with elevated PaCO2 during room air breathing at rest. Two weeks after CBD, the responses to elevated inspired CO2 had returned to normal. Thus, although the carotid chemoreceptors may normally play a role in the hypercapnic response, their loss may be compensated for by changes occurring elsewhere in the ventilatory control system.

Data on the ventilation vs. metabolic rate relation during exercise and the temporal pattern of PaCO2 during exercise are consistent with previous studies on the effect of CBD on the exercise hyperpnea in adult goats and ponies (2, 18-20), which showed that the carotid bodies function as "fine tuners" of the ventilatory response to exercise.

Somjen (24) postulated that the exercise hyperpnea (as well as autonomic regulation in general) was a "learned response" that developed postnatally. Data from the present study indicate that carotid chemoreceptors are not necessary for the development of normal ventilatory responses to exercise in neonatal goats. However, because the nCBD goats appeared to have normal ventilatory responses to hypoxia and hypercapnia, it is possible that feedback from other sensors may have guided the development of the exercise hyperpnea. Therefore, the data neither support nor refute Somjen's hypothesis.

Resting cardiovascular measurements. Other studies have noted an increase in MAP and/or HR after CBD, but these observations are from (relatively) acute CBD neonates (5, 7). Chronic elevation of MAP is not considered to be a consequence of CBD (6). The results of the present study indicate that nCBD does not result in long-lasting, dramatic changes in blood pressure or HR.

HR significantly decreased with age in both intact and nCBD goats, indicating that carotid baroreceptors are not necessary for this commonly observed phenomenon.

In a previous study on blood pressure and HR regulation in adult CBD ponies, regulation of MAP and HR during hypoxia was followed for 4 yr postsurgery. The ability to regulate cardiovascular function returned with the same time course as peripheral chemosensitivity and was similarly partially compromised by aortic nerve section 4 yr after the initial denervation (1).

Studies on the development of neural systems. Studies in other afferent neural systems, such as the visual system, have revealed that some neural connections (and therefore function) develop in the postnatal period (12, 25). In addition, it has been shown that the postnatal development of these connections is dependent on neural activity within the developing system. In addition, the effects have been shown to extend through several synapses, implying that silence (dysfunction) in one neural path could have a widespread effect on a developing system. It is important to note that the most pronounced effects are observed in young animals in which neural connections are not yet fully formed.

It is also important to note that the above results were obtained in the visual system, which does not have extensive interconnections among various nuclei and/or cell groups. Information from receptors located in the right or left eye (or retinal hemisphere) is kept separate until the visual cortex, and there are no extensive interconnections between cells and paths proximal to the visual cortex. In anatomic terms, specific areas of the retina are connected to specific areas of the lateral geniculate nucleus, which in turn are connected to specific areas of the visual cortex. A deficit (silence) in one location of the system is "translated" into a deficit (silence) in other locations.

This arrangement contrasts with the respiratory control system, which exhibits extensive interconnections among respiratory nuclei as well as redundant afferent systems (22). The consequence of neural silencing in highly interconnected networks is less predictable. Activity in other parts of the system (redundant sensors) could be increased to replace the lost input. Alternatively, input from a relatively minor source could become the driving influence. In either case, the concept of a "plastic network" is consistent with the effects observed in (multiple) denervation studies (20). The design of the system helps maintain the output of the respiratory control network. The absence of permanent changes in ventilatory control with CBD at 1-3 days of age could be because of fundamental differences between ventilatory control neural networks and neural networks in the visual system. Alternatively, absence of permanent changes in the present study could be related to the age of CBD, i.e., CBD may not have been at an age (critical window) at which attenuation of afferents causes permanent changes.

Summary

The data of this study showed that CBD at 1-3 days of age in goats does not lead to permanent changes in breathing greater than those observed after CBD in adult goats. The respiratory control system of neonatal goats appears highly plastic and appears to recover peripheral chemoreceptor function after CBD, probably at a site other than the carotid bifurcation. The recovered peripheral chemoreception was sufficient to allow normal responses to hypoxia, hypercapnia, and exercise, indicating that respiratory control had not been compromised. The low mortality may be related to the high degree of plasticity observed in goats. Only one of the six CBD goats exhibited severe breathing irregularity of the type reported by previous investigators. This irregularity disappeared within the first month postsurgery, and this was not the goat that died, indicating that if absence of carotid afferents is indeed a cause of death, it is not necessarily associated with irregular breathing.


    ACKNOWLEDGEMENTS

This study was supported by the Sudden Infant Death Research Fund of Wisconsin, by National Heart, Lung, and Blood Institute Grant HL-25739, and by Veterans Affairs.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. V. Forster, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.

Received 22 October 1998; accepted in final form 16 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 87(3):1026-1034
8570-7587/99 $5.00 Copyright © 1999 the American Physiological Society


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