INTRODUCTION

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THE CAROTID BODY IS A MAJOR peripheral chemoreceptor in mammals. Its afferents provide a tonic excitatory input to medullary neurons relayed mainly via the ventrolateral nucleus of the solitary tract (NTS_VL) (7) to maintain eupneic breathing (8). It also mediates increased breathing during acute and chronic hypoxia (9). It is essential for chemoreception in certain periods of postnatal development of the respiratory control system (5, 6). Thus several studies have utilized carotid body denervation (CBD) in neonatal animals as a model for studying sudden infant death syndrome (SIDS) (4-6, 42, 43).

The pre-Bötzinger complex (PBC) is postulated as the center of respiratory rhythmogenesis (11, 33, 35, 47). Although direct connections between the PBC and NTS_VL have not been proven, studies have show that NTS_VL projects to the ventrolateral medulla, including the region now called "PBC" (27, 39). The PBC contains a high percentage of propriobulbar neurons that project to the bulbospinal premotor respiratory neurons (2). Our previous studies showed that cytochrome oxidase (CO) activity in the PBC exhibited a plateau at postnatal days (P) 3-4 and a distinct, transient decrease at P12, despite a general increase in CO activity with age (17, 18). Coincidentally, a decrease in glutamate (Glu) and N-methyl-D-aspartate receptor subunit 1 and an increase in GABA, GABA_B receptor, glycine receptor (GlyR), and -amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit 2 were observed in PBC at P3-4 and P12 (18). Our data suggest that there are two vulnerable windows in the rat's postnatal development, at P3-4 and especially at P12, during which the animal may be more susceptible to the detrimental effects of respiratory distress, such as CBD. However, few studies have been done to show the effects of CBD on the postnatal development of PBC.

The present study was aimed at testing our hypothesis that the PBC would be more affected by CBD at the two vulnerable windows than at other times. CO was used as a marker of the neurons' metabolic capacity, which, in turn, reflects their level of functional activity (49, 50). Neurokinin-1 receptor (NK1R) is enriched in the PBC and was used to localize the PBC in the ventrolateral medulla (12).

	MATERIALS AND METHODS

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Tissue preparation. A total of 140 Sprague-Dawley rats from 10 litters were used in accordance with the National Institutes of Health and the Medical College of Wisconsin regulations. Litter sizes were 12-16 pups, and, at every time point studied, five pairs (CBD and sham) of rats from five different litters were used. CBD consisted of a bilateral retromandibular incision in the neck, removal of the carotid bodies, and section of the carotid sinus nerves. Sham surgery was performed with the same incision but no CBD. CBD and sham surgery were done on pairs of animals at each postnatal day from ages P2-P14 and on P21. For ages P2-P6, pups were anesthetized with ice (hypothermia), whereas for ages P7-P14 and P21, pups were anesthetized with intramuscular injections of ketamine (40 mg/kg), xylazine (2.5 mg/kg), and acepromazine (0.6 mg/kg) (Phoenix Laboratories, St. Joseph, MO). After recovery, rats were returned to their respective dams. Three days after surgery, rats were anesthetized with 4% chloral hydrate (0.1 ml/10 g ip, Fisher Scientific, Fair Lawn, NJ) and perfused through the aorta with 4% paraformaldehyde in 0.1 M PBS, pH 7.4, and 4% sucrose. The brain stems were removed and postfixed by immersion in the same fixative for 3 h at 4°C. They were then cryoprotected in increasing concentrations of sucrose (10, 20, 30%) in 0.1 M PBS at 4°C, frozen on dry ice, and stored at 80°C until use.

CO histochemistry. Coronal sections of frozen brain stems were cut at 12-µm thickness with a cryostat. At each time point, three sets of serial sections from each pair of CBD and sham rats were mounted on the same gelatin-coated slides and processed for CO histochemistry, NK1R immunohistochemistry, and Nissl staining, respectively. For CO histochemistry, the sections were incubated in 0.05% 3,3'-diaminobenzidine (Sigma Chemical, St. Louis, MO), 0.02% cytochrome c (type III, Sigma Chemical), and 4% sucrose in 0.1 M PBS (pH 7.4) at 37°C in the dark for 3 h (48). After incubation, the sections were washed with cold 0.1 M PBS (pH 7.4) three times, 5 min each. The slides were then air dried and coverslipped.

Immunohistochemistry. Coronal sections of frozen brain stems were obtained as described above. Sections were blocked overnight at 4°C with 5% nonfat dry milk, 5% normal goat serum, and 1% Triton X-100 in 0.1 M PBS (pH 7.4). They were then incubated at 4°C for 36-48 h in the primary antibody against NK1R (Sigma Chemical) diluted at 1:10,000 in the same solution as used for blocking. Sections were then incubated in the goat anti-rabbit IgG-horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA) at 1:100 dilution in the modified blocking solution (without Triton X-100) for 4 h at room temperature. Immunoreactivity was detected with 0.05% 3,3'-diaminobenzidine-0.004% H₂O₂ in PBS (pH 7.4) for 5-10 min, and the reaction was stopped with cold PBS (pH 7.4). The sections were washed with cold 0.1 M PBS (pH 7.4) three times, dehydrated, and coverslipped.

Cell area measurement. The long and short axes of neuronal cell bodies in the PBC were measured with a reticule and a ×40 objective lens in cresyl violet-stained sections. Between 150 and 200 neurons were measured for each age group. The average diameter was calculated, and the cell areas were determined.

Quantitative optical densitometry. Optical densitometric (OD) measurements of reaction product of CO were performed with a Zeiss Zonax MPM 03 photometer, a ×25 objective, and a 2-µm-diameter measuring spot. White (tungsten) light was used for illumination, and all lighting conditions were held constant for all of the measurements. The white matter was used as an internal standard for measurements because of its very low levels of CO activity. Thus the white matter was set at zero for each section measured. The OD value of each neuron in the PBC was an average reading of two to four spots in the cytoplasm. Thirty to eighty neurons in the PBC for each rat and a total of 150-350 neurons at each age were measured. The mean OD value and standard deviations of reaction product of CO at each age were calculated. Statistical comparisons were made between CBD and sham by using both one-way ANOVA and Student's t-test. Developmental trends within each group (sham, CBD, or normal) were analyzed by the Tukey test, with comparisons being carried out between each successive pair of time points. Significance was set at P < 0.01 for one-way ANOVA and P < 0.05 for the t-test and Tukey test.

	RESULTS

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General condition of animals after surgery. Compared with sham rats of the same age, CBD rats showed lower body weight (Fig. 1), lower body temperature measured rectally, and fewer and slower movements and were visibly weaker; and less milk and food intake was observed (P7 or older were better than younger ages). Body weights of the sham group were comparable to those of normal animals at every age examined (data not shown). The mortality in our study was lower than that of other studies (42). In the CBD group, only one rat died 2 days after surgery at P4 (1/6), and two died 2 and 3 days after surgery at P7 (2/7), respectively, whereas no sham rats died after surgery. The main reason of death after surgery in these three CBD rats appeared to be nutritional deficiency observed from their decline in nursing. They and their sham counterparts were not included in the final analysis of 140 animals.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Body weights of rats in carotid body denervation (CBD) and sham groups 3 days after surgeries. Values are means ± SD. CBD rats had significantly lower body weights than sham rats at all postnatal ages examined: *** P < 0.001.

NK1R-immunoreactive neurons in the PBC. In general, the expression of NK1R in the PBC of the sham and CBD group was similar to that described previously (18).

CO-reactive neurons in the PBC. CO-reactive neurons in the PBC exhibited dark, moderate, or light intensities of CO labeling, with processes traceable for short distances in different directions. In general, CO activity in both CBD and sham groups exhibited a gradual increase with age. OD measurements of CO reaction products could also be subdivided into three main levels of CO activity (high, moderate, and low) (Fig. 2). Both CBD and sham groups showed similar developmental trends, although there were some differences in the time course among them. However, CBD rats had lower levels of CO activity than that of sham rats, except when CBD was done on P12 or P13. When CBD was performed on P3, P9, or P11, OD values in the CBD group were only 82.4, 82.0, and 82.0% of sham values among neurons of high-CO activity; 80.8, 81.8, and 80.0% of sham values in neurons of moderate CO activity; and 79.7, 82.4, and 76.8%, respectively, of sham values in neurons of low-CO activity (Figs. 2 and 3).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Optical densitometric measurements of cytochrome oxidase (CO) reaction product in the pre-Bötzinger complex (PBC) of CBD and sham groups 3 days after surgeries. Values are means ± SD. PBC neurons with high (A), moderate (B), and low CO activity (C) in CBD rats showed significantly lower optical densitometric values than those of sham rats at postnatal days (P) 6, P8, P10-P14, P17, and P24 (surgeries were performed on P3, P5, P7-P11, P14, and P21, respectively); only at P16 (surgeries were performed on P13) did CBD rats show higher optical density values for all 3 levels of CO activity than those of sham rats: * P < 0.05, ** P < 0.01, *** P < 0.001.

View larger version (91K): [in this window] [in a new window]   Fig. 3.   Neurons in the PBC histochemically reacted for CO. A, C, E, G, and I: neurons from sham-operated animals were perfused at P6, P12, P13, P14, and P24, and surgeries (S) were done 3 days earlier, at P3, P9, P10, P11, and P21 (S3, S9, S10, S11, and S21), respectively. B, D, F, H, and J: neurons from CBD animals with surgeries and perfusion done at the same ages, respectively. CBD rats showed lower CO activity at P6, P12-P14, and P24 compared with sham rats. Note that sham rats exhibited a dramatic decrease in CO activity at P13 and an increase again at P14. However, CBD rats did not show an increase in CO activity at P14 after a dramatic decrease at P13, indicating that there was a delay in the development of the CO pattern in CBD rats compared with sham rats. Scale bars: 40 µm.

Developmental trend of CO activity in sham and CBD animals. CO activity in the sham group showed a first peak at P6 (surgery at P3), a plateau at P7-P9, a second peak at P12, a dramatic drop at P13, followed by an increase at P14, another drop at P15, and a gradual increase until P24. CO activity in the CBD group, however, did not show the first peak until P7, a plateau at P9, a much smaller peak than that of sham at P12, a dramatic decrease at P13 for 2 days, then an increase at P15 (1 day later than sham rats) for 2 days, another decrease at P17, followed by an increase at P24 (Figs. 2-4).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Optical densitometric measurements of CO reaction product in the PBC of CBD and sham rats (A) as well as in normal rats [summarized from our previous study (18); B]. Data represent averages of optical density values of high, moderate, and low CO-reactive neurons in each of the 3 groups (18). See text for details. Values are means ± SD. Student's t-test was used for comparison of sham and CBD group at each time point: * P < 0.05, ** P < 0.01, *** P < 0.001. Tukey's test was used for the trend within each group: sham, CBD, and normal. Comparisons were made between 2 consecutive time points, and the level of significance was marked on the later time point: + P < 0.05, ++ P < 0.01, +++ P < 0.001. Note that the decrease at P7 is only significant when the 3 metabolic groups are pooled together (B). No significance was found when the groups were analyzed separately (18). Note also that comparisons between our previous and the present study should only be made with respect to trends rather than the absolute optical density values, the latter being relative numbers that can vary with photometer setting. For each study, however, all settings were kept constant.

Neuronal area measurements. Somal sizes of PBC neurons at each developmental age of both CBD and sham groups are shown in Fig. 5. Somal sizes of the sham group at every age examined were comparable with those of our previous study (18). Somal sizes of the CBD group were significantly smaller than those of the sham group when CBD was performed on P2-P7. However, there was no significant difference in somal sizes between CBD and sham groups when surgeries were performed on P8 or later (Fig. 5).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Somal area in the PBC of CBD and sham rats 3 days after surgeries. Values are means ± SD. At P5-P10 (surgeries were performed on P2-P7, respectively), CBD rats showed significantly smaller somal areas in the PBC than those of sham rats: *** P < 0.001.

	DISCUSSION

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Four major findings of the present study are as follows. 1) CBD animals suffered a significant loss in body weight compared with sham animals at all ages examined. This implies that CBD affects the general development of the rat for the first 3 postnatal wk, including the development of the respiratory control system and other regions of the central nervous system. 2) When CBD was performed on P2-P7, the somal size of PBC neurons was significantly smaller than that of sham animals. This difference was not seen when CBD was done on P8 or later, suggesting that P2-P7 is a sensitive window for somal size development in the PBC. 3) The level of CO in the PBC of CBD animals was lower than that of sham animals at most ages examined, except when CBD was performed on P12 and P13 (Fig. 4A; see below). The level of CO in CBD animals also exhibited a rather flat pattern compared with those of sham and normal animals (Fig. 4), indicating that CBD had a detrimental effect on the metabolic integrity and possibly neuronal functioning of PBC neurons. 4) The prominent rise-fall-rise pattern seen in normal animals between P11 and P13 (18) is retained in both sham and CBD animals (compare Fig. 4A with Fig. 4B). However, sham animals showed a 1-day delay in this maturational process, with the rise-fall-rise pattern occurring at P12-P14 instead. CBD animals exhibited a 1- to 2-day delay, with the rise at P12, fall at P13 and P14, and rise again at P15 and P16. The retention of such a pattern, although delayed and prolonged, strongly suggests that it is a maturational process that is largely genetically programmed.

What could be the cause of such a pattern? Previously, our laboratory has shown that a higher level of CO activity correlates directly with a greater proportion of excitatory inputs, whereas a predominance of inhibitory inputs is associated with a lower level of CO activity (23, 25, 26, 49). This is related to the fact that the bulk of energy consumed by neurons is used for repolarizing their membranes after depolarization, whereas repolarization after inhibitory hyperpolarization is mainly passive (49). CO activity in neurons is also correlated with their sustained firing rate generated synaptically or spontaneously (50). In the PBC, the plateau at P3-P4 and a drastic drop in CO activity at P12 are correlated with a concomitant decrease in immunoreactivity of Glu and N-methyl-D-aspartate receptor and a rise in GABA, GABA_B receptor, and GlyR at those times, with the change at P12 being more prominent than that at P3-P4 (18). Thus the maturational process in the PBC imposes a stronger inhibitory drive than an excitatory one, especially at P12. We proposed that this critical period might render the animal less able to overcome the detrimental effect of respiratory insults. In the present study, CBD induced a delay and a prolongation of this maturational process. Superimposed on an overall reduction in CO activity in CBD animals, the 3-day rise-fall-rise pattern around P11-P13 in normal animals is retained but delayed and stretched out to a 5-day pattern at P12-P16 (Fig. 4). This delay and prolongation resulted in apparent equal or higher values of CO in CBD animals than those of sham at P15 and P16, when CBD was performed at P12 and P13, respectively. If a delay did not occur, then CO activity at P15 and P16 would have been lower than that of the sham group at P14 (which normally occurs at P13; Fig. 4). Thus, despite the trauma of CBD, PBC neurons still proceeded with a general progression of synaptic maturation, synaptic imbalance, and synaptic adjustment that is likely to be genetically determined.

Sham animals also exhibited a delay in their metabolic development, most pronounced when CBD was done between P8 and P13. It is possible that the trauma of anesthesia and surgery induced a 1-day delay in the progression of CO development from P11 to P16 compared with that in normal animals (Fig. 4). Despite this delay, the progression of rise-fall-rise pattern at P11-P13 in normal animals is retained at P12-P14 in sham animals. Again, the process of synaptic maturation was able to proceed, although with a 1-day delay, in these animals.

Respiratory rhythm generation may undergo a developmental transformation. Rhythm generation in the neonate is largely pacemaker driven (presumably in the PBC), but network inhibitory interactions become more important in the respiratory rhythm generation and control as development proceeds (46). PBC neurons in vivo in the adult (36, 40) exhibit larger inhibitory hyperpolarizations than in the neonatal in vitro system (3, 34, 44). Disruption of rhythmic activity by the blockade of synaptic inhibition both in vivo and in vitro indicates that the system is not mature until P15 (29, 30). Our data showed that the CO pattern in the normal PBC was mostly mature at P13 but exhibited increases even after P17. The receptors of major inhibitory transmitters, GABA and glycine, undergo a functional shift from excitatory to inhibitory (37, 51) and a subunit switch from ₂ to ₁ (for GABA_A receptors) and from ₂ to ₁ (for GlyR) (10, 13, 32). Perhaps the subunit switch of inhibitory receptors coincides with heightened inhibitory circuits after a peak in excitatory circuits. The period of major synaptic adjustment appears to occur at P11-P13 in normal animals, at P12-P14 in sham animals, and at P12-P16 in the CBD group.

In addition to surgery, CBD animals also suffered a loss of a key peripheral chemoreceptor. Previous reports indicated that CBD can induce transient hypoventilation (9, 19, 20, 28, 38, 43), lower response to hypoxia (20, 42), a lack of secondary decline in response to hypoxia (22), and lower body weight compared with sham animals (42). The removal of signals from the carotid body to the NTS_VL would reduce the functional activity of the NTS_VL, leading to lower excitatory afferent drive from the NTS_VL to the PBC. This, in turn, results in decreased CO activity in the PBC, as sensory deprivation does to other systems (23, 25, 26, 49). Our data showed that CBD induced a lower body weight at all ages examined 3 days after denervation. CBD also reduced the increase in somal size when performed between P2 and P7. CBD might induce mild hypoxia postoperatively when other peripheral chemoreceptors could not compensate sufficiently for the loss of carotid body afferent drives. Mild hypoxia could affect functioning of the entire body, including respiration as well as feeding.

Malnutrition or undernutrition from compromised feeding could induce an impairment in neuronal membrane dynamics and delay the functional and morphological maturation of neurons (14, 16, 45), resulting in dendritic arbor hypoplasia and mild reduction in somal size (31). Malnutrition possibly prolongs the period of DNA synthesis and cell cycle time, starting from P12 (16). Inadequate iron intake induces a deficit in myelination (24), and protein malnutrition causes lower body weight gain, although lower brain regional weight could be observed only on P15 (1). Moreover, malnutrition could induce lower physical growth as well as delay in ear opening and eye opening (31). Undernutrition could also alter some neural systems, such as the noradrenergic system (41), leading to functional abnormalities.

To rule out the possibility that CBD might cause a generalized decrease in CO activity, we also examined other brain stem nuclei, either related or unrelated to respiration. Our preliminary data indicate that these nuclei do not respond alike to the same insult, namely CBD.

The effects of CBD have been reported to be developmentally dependent, being greater when CBD is performed on P7-P8 in rats (42) and on P10-P25 in piglets (20). The present study extended the findings in the rat to a more detailed, day-to-day analysis of metabolic consequences of CBD in the PBC. In the present study, CBD appeared detrimental at most ages of animals examined. When CBD was done on P3, CO activity remained at a plateau 3 days later whereas that of sham animals exhibited a sharp increase (Fig. 4A). The delay and prolongation of the maturational process when CBD was done between P8 and P13 were discussed above. Thus the present study did not find a more detrimental effect of CBD at P7-P8. In terms of normal CO development, significant reduction at P7 was found only when we pooled all three metabolic cell groups together (Fig. 4B), but not when they were analyzed separately (18). The fluctuations in CO activity at P7-P9 were not nearly as dramatic as those at P11-P13. Similarly, there were no correlated neurochemical adjustments that could be linked to CO changes at P7-P9 as they could at P3-P4 and P11-P13 (18), and response to CBD or sham operations done at P7-P9 was not as distinct as those at P12 (Fig. 4A). Thus P7-P9 appears to represent a period of mild adjustment, whereas P12 (or P11-P13) is a period of major adjustment metabolically and neurochemically.

The effects of CBD have been shown to be physiologically compensated within 2 wk to 3 mo (19, 21, 28, 42). It is likely that other chemoreceptors, notably from the aortic arches, are able to compensate for the loss of carotid body input (42).

In conclusion, our study suggests the following: 1) There is an intrinsic developmental pattern of CO activity in the PBC of rats, with vulnerable windows occurring normally at P3-P4 and, most notably, around P12 (P11-P13). Certain conditions, such as CBD and sham surgery, modify the trend in extent and time course, but neither eliminate nor change the overall pattern. 2) CBD decreases CO activity in the PBC during postnatal development. 3) When the normal maturational process in the PBC is delayed and prolonged, especially around the critical period, it may render the system even less able to overcome the stresses of respiratory insults.

Ninety percent of SIDS occurs in the first 6 mo of life, with a peak at 2-4 mo, implying a vulnerable period in development. Kinney et al. (15) have proposed a triple-risk model for the pathogenesis of SIDS: a critical period of development, vulnerable infant, and exogenous stressor(s). Our previous study strongly suggests a critical period (or periods) of development (at least in the rat) (18). In the present study, CBD potentially renders the pups more vulnerable. However, a host of other intrinsic or extrinsic factors could contribute to the vulnerability of the infant, and herein lies the most difficult part of the "mystery" of SIDS and of a suitable "animal model" for SIDS. A full test of the validity of the triple-risk model necessitates simultaneous introduction of all three factors.