Main

Circulating GCs are thought to play an important role both in the adaptation of the neonate to extrauterine life in the immediate postnatal period(1, 2) as well as in the development of tissue function in later postnatal life(3–11). A well characterized and critical function of the GCs at the time of birth is the promotion of lung function via enhancement of tissue differentiation, surfactant synthesis and other effects(1, 2, 12). In support of this idea, plasma cortisol levels on postnatal d 2-6 have been inversely correlated with a requirement for surfactant, respiratory support, and inotropic medication, independent of gestational age at birth, in premature human neonates(13).

In addition to the immediate postnatal period, evidence from animal models indicates that GCs are important during a more extended period of early development. In a number of tissues (e.g. lung, intestine, pituitary, corticolimbic brain regions), GC receptor levels are highest or are present only during the first few weeks of life compared with later, suggesting that GCs may play a specific role at this time(10, 14). These early changes in GC receptor levels are associated with a distinct pattern of basal corticosterone secretion during this period. In the neonatal rat, plasma GC levels are relatively high at birth, fall to barely detectable levels by postnatal d 2, and begin to rise around d 14-15 to reach adult levels by d 21 after birth(10, 15, 16). It appears that human neonates show a similar developmental profile; cortisol levels have been demonstrated to be high at birth, but to fall below adult levels during the first few weeks of life in both full-term and premature human neonates(13, 17–20). These distinctive developmental patterns of circulating GCs and of GC receptors suggest that levels of circulating GCs may play a critical role in tissue development during this period. Such effects have been demonstrated. For example, alterations in levels of circulating GCs during the first postnatal weeks have been shown to alter neurogenesis and/or neuron death in rat cerebellum and hippocampal dentate gyrus(6–9), and to influence developmental induction of a wide range of hepatic and gastrointestinal enzymes(11).

In the human neonate, factors known to have important effects on GC levels in the immediate postnatal period are the route of birth and the presence of certain birth complications. Infants born by elective C-section(i.e. without labor) have been shown consistently to have significantly lower plasma cortisol levels at birth than do vaginally born infants (e.g.21–25). Together with this, an increased frequency of respiratory morbidity(respiratory distress syndrome type II) has been reported for term neonates born by elective C-section in comparison with vaginally born infants(26). On the other hand, in comparison with levels measured for control neonates matched for gestational age at birth, plasma cortisol levels have been reported to be high in both term and preterm infants born after intrauterine acidosis or other signs of perinatal hypoxia(18, 20, 27–30). This suggests that a stressful birth activates cortisol secretion, likely as an adaptive response to the birth conditions.

Although birth complications clearly affect circulating GC levels at birth, it is unclear whether birth condition might have more lasting effects on the developmental profile of basal GC secretion during the first few weeks of life. In the present study, we have used a rat model to test whether birth by C-section or an acute period of birth anoxia can affect the developmental profile of basal GC secretion in the first few weeks of life. The rat model has an advantage, in that one can maintain conditions of birth and of postnatal development uniform within an experimental group. However, insofar as parallels can be drawn between rat and human systems, the rat model of C-section birth likely relates more to the condition of the premature rather than the term human neonate(31). In addition to plasma levels of total corticosterone (the major circulating GC in the rat), the present study also measured plasma levels of CBG, the main plasma-binding protein for corticosterone. This allowed for the calculation of levels of free, i.e. biologically active, corticosterone, for testing whether measures of total corticosterone accurately reflect the dynamics of free corticosterone, under various conditions.

METHODS

C-section and intrauterine anoxia. For the C-section birth procedure, timed pregnant Sprague-Dawley rats (Charles River, St. Constant, Quebec) at 22 d of gestation (i.e. on the day of birth) were decapitated, an abdominal incision was made, the uterus was isolated from its blood supply and surrounding connective and fatty tissue (10-15 s), and the pups were rapidly delivered. The time between killing of the dam and delivery of the last pup in the C-section group was <1.5 min. Umbilical cords were ligated, and the animals were placed on a heating pad until given to their surrogate mothers (1 h). Survival was 100% in the C-section group. Rat pups underwent anoxia during C-section birth using procedures modified by Bjelke et al.(32). For this, the pregnant dam was decapitated, the intact uterus was isolated from its blood supply and other tissues, and an acute anoxic episode was induced by immersing the intact uterus into a 37°C saline bath for 15 min (C-section + anoxia group). The pups were then delivered and stimulated by gentle tapping until breathing became even (30-40 s). No other means of artificial resuscitation was used. The rat is able to sustain longer periods of anoxia at birth than is the human neonate(33). Because rats undergoing 15 min of birth anoxia began breathing without artificial resuscitation and exhibited normal behavior as adults [except under particular testing conditions; see Boksa et al.(34)], this period of anoxia in the rat may represent a mild to moderate, as opposed to severe, anoxic episode.[Although vital signs were not monitored in the present study, Bjelke et al.(32) reported that heart rate in similarly treated pups remained at 140-160 beats/min during the initial 5-7 min of anoxia before decreasing steadily to 80-100 beats/min after 15 min. The same authors found highly variable levels of oxygen saturation in anoxic pups; levels ranged from 15-50% to 10-35% after 5 and 15 min of anoxia, respectively.] Survival was 90-95% after 15 min of birth anoxia. Pups born vaginally served as controls (control group). Pups delivered by C-section were born from a total of seven dams, pups born by C-section with added anoxia were from nine dams, and vaginally born pups were taken from 16 dams. All pups, including vaginally born controls, were coded according to birth condition by s.c. injection of a small amount of India ink into a specified paw. To minimize differential rearing effects, pups of both sexes from all groups were cross-fostered in mixed litters (13-14 pups/dam) by each surrogate dam, until weaning at 21 d of age. A total of 16 surrogate dams were used; for each experimental group, in most cases one pup of each sex was taken from each litter to be killed at each of the indicated ages; in a minority of cases a maximum of two pups of the same sex were taken from a single litter. Animals were maintained on a 12 h:12 h light:dark schedule (lights on and off at 0800 h and 2000 h, respectively) with free access to food and water. All procedures with animals were performed in accordance with the guidelines of the Canadian Council on Animal Care and were approved by the McGill University Animal Care Committee.

Measurement of plasma corticosterone and corticosteroid binding globulin. Pups at the various ages indicated in the figures were quickly weighed and decapitated for trunk blood collection in EDTA-coated Eppendorf tubes. Samples designated “at birth” or “immediately after birth” were taken <5 min from the time of birth. Numbers of pups in each experimental group used for measurements at various ages are indicated in the legends to individual figures. Sacrifice and blood sampling were done between 1200 to 1600 h. Blood samples were centrifuged (1,000 ×g, 10 min, 4°C) and aliquots of the supernatant stored at-80°C. Body organs were dissected, and wet weight was recorded.

On the day of the corticosterone analysis, plasma samples were thawed on ice. Plasma corticosterone was measured by RIA as described by Krey et al.(35) using a highly specific antiserum (B3-163) from Endocrine Sciences (Tarzana, CA), [3H]corticosterone (88 Ci/mmol; Dupont NEN, Boston, MA) as tracer, and 1 μL of plasma. The antiserum cross-reacts slightly with desoxycorticosterone (≈4%), but not with cortisol (<1%), and the minimum level of detection for corticosterone was 10 pg/mL. The conditions of this assay allow for measurement of total (both bound and free) plasma corticosterone.

Plasma CBG was measured by a steroid-binding capacity assay as described by Meaney et al.(36). In brief, endogenous steroids were removed from plasma samples by passing the sample through a Sephadex LH-20 column. Aliquots of the plasma were then incubated in buffer containing a saturating concentration (80 nM) of [3H]corticosterone for 90 min at 2-4°C. Nonspecific binding was defined in parallel incubations containing a 200-fold excess of nonradioactive corticosterone. Separation of bound from free [3H]corticosterone was achieved using Sephadex LH-20 chromatography. Protein was determined using the method of Bradford(37).

Calculation of CBG-bound corticosterone, free corticosterone, and percent of total corticosterone bound to CBG. Using measured values for plasma total corticosterone and CBG, values for corticosterone bound to CBG were derived from the mass action equation described by Plymate et al.(38) and by Hanisch et al.(39), as follows: Equation where BCBG is the concentration of CBG-bound corticosterone;b is 1/k + CBG + B; k (76 × 106 M-1) is the association constant of corticosterone and CBG; CBG is the concentration of plasma CBG; B is the concentration of plasma total corticosterone; and a is B × CBG. [Use of this calculation across developmental ages in the rat makes the assumption that the association constant for corticosterone and CBG is similar in rats of various ages. Savu et al.(40), using Scatchard analyses, have reported similar values for the association constant for plasma CBG and corticosterone measured in rat fetuses at 19 d of gestation, their pregnant mothers and adult, non-pregnant rats. The assumption that the affinity of CBG for corticosterone does not change with age in the rat is further supported by the strong correlation between CBG measured by RIA and CBG measured by steroid binding capacity in male and female rats from 1 to 12 wk of age(41).] The concentration of free corticosterone in plasma was calculated as [total corticosterone] - [corticosterone bound to CBG]. The percent of total corticosterone bound to CBG was calculated as(corticosterone bound to CBG/total corticosterone) × 100.

Statistical analysis. Data were analyzed using two-way analysis of variance on log-transformed data to correct for heterogeneous variance; in cases where some of the observations were zero or small numbers (plasma total corticosterone, free corticosterone, CBG, percent corticosterone bound to CBG) the transformation log (x + 1) was used. Individual differences were evaluated by post hoc Neuman-Keul's tests, where appropriate.

RESULTS

Males

Figure 1a shows plasma levels of total corticosterone measured at various ages after birth in male rats born vaginally, by C-section, or by C-section with 15 min of added anoxia. Two-way analysis of variance showed a significant interaction between birth group and age[F(10, 120) = 5.02, p < 0.0001] and a significant overall effect of age [F(5, 120) = 38.25, p < 0.0001], but no group effect over all ages F(2, 24) = 0.26, p = 0.48].

Figure 1
figure 1

Plasma total corticosterone (a) and free corticosterone (b) in male rats taken at various times after vaginal birth, birth by C-section or birth by C-section with anoxia. Animals born vaginally, by C-section, or by C-section with 15 min of added anoxia(C + Anoxia) were decapitated immediately after birth(Birth) or at the indicated times after birth, and total corticosterone in a sample of trunk blood was measured. Free corticosterone was calculated from measures of plasma total corticosterone and CBG, as described in “Methods.” Results at each time point are mean± SEM values. For vaginally born animals, numbers of animals taken at birth, at 1 h after birth, and at 1, 7, 14, and 35 d of age, respectively, were 17, 12, 11, 15, 16, and 11; corresponding n's for animals born by C-section were 10, 9, 8, 9, 9, and 9; and for animals born by C-section with added anoxia were 8, 8, 9, 10, 10, and 8. Symbols above bars denote values significantly different from vaginal birth at p < 0.01(**) and p < 0.05 (*) and significantly different from C + Anoxia at p < 0.05 (Ψ).

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The pattern of plasma total corticosterone levels measured over time in vaginally born male controls (Fig. 1a) is consistent with that previously reported in the literature. In these vaginally born males, moderate levels of total corticosterone were measured at birth, and this level was maintained at 1 h and 1 d after birth. Total corticosterone fell to low levels at d 7 and was maintained at low levels on d 14 in the vaginally born males; however, by 35 d of age, total corticosterone reached higher levels, characteristic of the adult animal.

C-section versus vaginal birth . Post hoc Neuman-Keul's tests showed that, immediately after birth, male pups born by C-section had similar levels of plasma total corticosterone as did vaginally born males (Fig. 1a). However, at 1 h (31% of control;p < 0.01) and at 7 d (61% of control; p < 0.01) after birth, total corticosterone was significantly lower in the C-sectioned males compared with vaginally born controls. By contrast, plasma total corticosterone levels at 14 d of age were significantly higher in C-sectioned males (239% of control; p < 0.05) in comparison with vaginally born control males. At 35 d of age, total corticosterone levels in C-sectioned and vaginally born males were similar.

Table 1A shows plasma CBG activity measured at various ages after birth in male rats born vaginally, by C-section, or by C-section with 15 min of added anoxia. Two-way analysis of variance indicated a significant interaction between birth group and age [F(10, 115) = 4.64, p < 0.0001)] and significant effects of both age[F(5, 115) = 173.52, p < 0.0001)] and group[F(2, 23) = 8.73, p < 0.0015). In vaginally born male rats, CBG levels were maintained at 1 h after birth, fell by 1 d after birth, and reached very low levels at 7 d of age. However, CBG levels began to rise again by 14 d of age to reach adult levels by 35 d. At birth, plasma CBG levels in C-sectioned males were similar to those measured in vaginally born controls. One hour after birth, CBG levels were significantly lower (51% of control; p < 0.01) in the C-sectioned group in comparison with vaginally born controls. There were no significant differences in plasma CBG levels between C-sectioned and vaginally born males at 1, 7, 14, and 35 d of age. The percent of plasma total corticosterone that was bound to CBG (Table 1B) was calculated for male rats born vaginally, by C-section, or by C-section with added anoxia, using the measured values for plasma total corticosterone and CBG activity. Two-way analysis of variance indicated a significant effect of age [F(5, 120) = 58.31,p < 0.0001)] on this measure but no effect of group[F(2, 24) = 0.10, p = 0.91) and no significant interaction between birth group and age [F(10, 120) = 1.54, p = 0.13)]. In vaginally born control males, the percent of total corticosterone bound to CBG remained constant from birth to 1 d of age, fell to a very low value at 7 d, began to rise at 14 d, and reached its highest value at 35 d of age. The percent of total corticosterone bound to CBG was similar for C-sectioned and vaginally born male rats at all time points.

Table 1 Plasma levels of CBG (A) and calculated percentage of total plasma corticosterone bound to CBG (B) for male rats taken at various times after vaginal birth, birth by C-section, or birth by C-section with anoxia
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Plasma free corticosterone (Fig. 1b) was also calculated for male rats born vaginally, by C-section, or by C-section with added anoxia, using the measured values for plasma total corticosterone and CBG. Two-way analysis of variance showed a significant interaction between birth group and age [F(19, 120) = 4.04, p < 0.0001)] and a significant overall effect of age [F(5, 120) = 5.12,p < 0.0003)], but no group effect over all ages [F(2, 24) = 0.32, p = 0.73]. The pattern of changes in free corticosterone induced by C-section was identical to the pattern reported for total corticosterone. That is, free corticosterone levels were significantly reduced at 1 h (p < 0.01) and at 7 d (p < 0.05) after birth and were significantly elevated at 14 d (p < 0.05) in the C-sectioned animals, in comparison with vaginally born controls.

Male rats born by C-section showed no significant difference in body weight in comparison with vaginally born controls at birth and at 1 h after birth(Table 2). However, at both 1 and 7 d of age, C-sectioned males weighed significantly less than did vaginally born controls; this was reflected in reduced weight of all organs weighed (brain, adrenals, liver, kidney, spleen) on d 1 and reduced weight in kidney and spleen on d 7 in the C-sectioned animals. By d 14 (and d 35) total body weight in C-sectioned males was again similar to that in the vaginally born control group; the kidney was the only body organ observed to be of reduced weight on d 14 in the C-sectioned group.

Table 2 Body and organ weights of male rats taken at various times after vaginal birth, birth by C-section, or birth by C-section with anoxia
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C-section plus anoxia versus vaginal birth. At birth, male rats born by C-section with 15 min of added anoxia showed similar levels of plasma total corticosterone in comparison with vaginally born males (Fig. 1a). Total corticosterone was reduced at 1 h in the anoxic group (58% of control; p < 0.05) compared with control values, but was not significantly different from control on d 1 and 7. However, at 14 d of age, total corticosterone levels were significantly increased in the anoxic males (227% of control; p < 0.05) in comparison with vaginally born control males. At 35 d of age plasma corticosterone levels were again similar in anoxic and control males. Plasma CBG levels were significantly higher at 35 d of age in males born by C-section with anoxia (p < 0.05) compared with vaginally born males (Table 1A). Birth by C-section with anoxia had no effects on CBG levels at other time points (Table 1A), and there were no significant differences in percent of total corticosterone bound to CBG between the anoxic and the vaginally born groups (Table 1B). For male rats born by C-section with anoxia, the findings for total corticosterone were also reflected in values for free corticosterone. That is there was a significant reduction in free corticosterone at 1 h (p< 0.05) and a significant increase in free corticosterone at 14 d of age(p < 0.05), in males born by C-section with anoxia in comparison with vaginally born males (Fig. 1b).

Groups of male rats born by C-section with 15 min of added anoxia weighed less than did vaginally born males at birth and at 1 h, 1 d, and 7 d of age(Table 2). However, total body weight in the anoxic group was similar to that of vaginally born male controls by d 14 and 35 after birth. Weights of the spleen and kidney appeared to most closely reflect the reductions in total body weight, whereas brain and liver weights were better maintained.

Females

Figure 2a shows plasma levels of total corticosterone measured at various ages after birth in female rats born vaginally, by C-section, or by C-section with 15 min of added anoxia. Two-way analysis of variance showed a significant interaction between birth group and age[F(10, 85) = 3.20, p < 0.0016] and a significant overall effect of age [F(5, 85) = 28.28, p < 0.0001], but no group effect over all ages [F(2, 17) = 1.22, p = 0.32].

Figure 2
figure 2

Plasma total corticosterone (a) and free corticosterone (b) in female rats taken at various times after vaginal birth, birth by C-section, or birth by C-section with anoxia. See legend to Figure 1 for experimental details. Results are mean ± SEM values. For vaginally born animals, numbers of animals taken at birth, at 1 h after birth, and at 1, 7, 14, and 35 d of age, respectively, were 13, 11, 11, 14, 13, and 6; corresponding n's for animals born by C-section were 10, 7, 8, 8, 9, and 8; and for animals born by C-section with added anoxia were 8, 7, 9, 9, 9, and 8. Symbols above bars denote values significantly different from vaginal birth at p < 0.05 (*) and significantly different from C + Anoxia at p < 0.05 (Ψ).

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C-section versus vaginal birth. Findings in C-sectioned females were similar, in most respects, to the observations made for males.Post hoc Neuman-Keul's tests showed that there was no significant difference in plasma total corticosterone between C-sectioned and vaginally born females immediately after birth (Fig. 2a). However, total corticosterone levels were significantly lower in C-sectioned females at 1 h (45% of control; p < 0.05) and at 7 d (55% of control;p < 0.05) of age. By 35 d of age total corticosterone levels were similar in the C-sectioned and vaginally born groups of females.

Table 3A shows plasma CBG activity measured at various ages after birth in female rats born vaginally, by C-section, or by C-section with 15 min of added anoxia. Two-way analysis of variance indicated a significant interaction between birth group and age [F(10, 90) = 2.69, p < 0.0062)] and significant effects of both age[F(5, 90) = 197.24, p < 0.0001)] and group[F(2, 18) = 5.16, p < 0.017). Plasma CBG was significantly lower in C-sectioned females at 1 h (50% of control; p< 0.01) and also at 35 d (61% of control; p < 0.05) of age, in comparison with levels measured in vaginally born females (Table 3A). There were no significant differences in CBG levels between C-sectioned and vaginally born female control groups at other sampling times. Additionally, there were no significant differences in percent corticosterone bound between vaginally born and C-sectioned females (Table 3B); two-way analysis of variance indicated a significant effect of age [F(5, 85) = 35.36, p < 0.0001)] on this measure but no effect of group [F(2, 17) = 0.15,p = 0.86) and no significant interaction between birth group and age[F(10, 85) = 1.42, p = 0.19)].

Table 3 Plasma levels of CBG (A) and calculated percentage of total plasma corticosterone bound to CBG (B) for female rats taken at various times after vaginal birth, birth by C-section, or birth by C-section with anoxia
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Figure 2b shows plasma free corticosterone, calculated for female rats born vaginally, by C-section, or by C-section with added anoxia, using the measured values for plasma total corticosterone and CBG. Two-way analysis of variance showed a significant interaction between birth group and age [F(10, 90) = 2.30, p < 0.0185)] and a significant overall effect of age [F(5, 90) = 5.79, p < 0.0001)], but no group effect over all ages [F(2, 18) = 0.72,p = 0.50]. Similar to results for total corticosterone, female rats born by C-section showed a significant reduction in free corticosterone at 1 h(p < 0.05) and at 7 d of age (p < 0.05), in comparison with vaginally born females. In addition, C-sectioned female rats showed a significant increase in free corticosterone (Fig. 2b) at birth, in comparison with vaginally born controls, although this finding was not reflected in the total corticosterone measurements (Fig. 2a).

Females pups born by C-section weighed less than did the vaginally born group at birth and at 1 d of age; this was reflected only in a reduced kidney weight at birth and in a reduced liver weight on d 1 (Table 4). C-sectioned female rats showed no differences in total body or organ weights in comparison with vaginally born females when weighed on d 7, 14, or 35.

Table 4 Body and organ weights of female rats taken at various times after vaginal birth, birth by C-section, or birth by C-section with anoxia
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C-section plus anoxia versus vaginal birth. Female rats born by C-section with 15 min of added anoxia had similar levels of plasma total corticosterone at birth and at 1 h in comparison with vaginally born females (Fig. 2a). Total corticosterone was significantly higher on d 1 (263% of control; p < 0.05) but lower on d 7 (62% of control; p < 0.05) in the anoxic group of females in comparison with vaginally born females. There were no significant differences in total corticosterone levels between anoxic and vaginally born females, at 14 and 35 d of age. Anoxic females showed no significant alterations in plasma CBG, or in percent of total corticosterone bound to CBG, in comparison with vaginally born females, at any time point (Table 3, A and B). Similar to findings for total corticosterone (Fig. 2a), free corticosterone was higher on d 1 but lower on d 7, in anoxic compared with vaginally born female rats (Fig. 2b).

In females born by C-section with 15 min added anoxia, body weight was less than that of vaginally born controls in groups weighed at birth and at 1 d after birth (Table 4), but returned to control levels at later time points.

Comparison of Results for Males versus Females

Results for males and females were compared using two-way analysis of variance with sex and age as main factors, for each birth group (vaginal, C-section, and C-section plus anoxia); measures analyzed were plasma total corticosterone (Figs. 1a and 2a), free corticosterone (Figs. 1b and 2b), CBG (Tables 1A and 3A), and percent of total corticosterone bound to CBG (Tables 1B and 3B). For all measures, a significant effect of age (p < 0.0001), but no significant effect of sex was observed. No significant sex × age interactions were observed except for measures of CBG. There was a significant sex × age interaction for CBG measured in vaginally born [F(5, 75) = 4.09, p < 0.0024] and in C-sectioned animals [F(5, 60) = 3.25, p< 0.0117]; post hoc Neuman-Keul's tests indicated that plasma CBG levels differed between vaginally born males and females at 35 d of age(p < 0.01) and between C-sectioned males and females also at 35 d of age (p < 0.05).

DISCUSSION

The main finding of this study is that birth by C-section or by C-section with added anoxia can alter the early developmental profile of plasma corticosterone in comparison with that observed in a vaginally born animal; these alterations outlast the immediate postnatal period and can be detected from 1 h up until 14 d after birth in the rat. The most pronounced changes observed in this study were produced by C-section birth (without additional anoxia) in the male, although findings in the female followed a similar pattern. Immediately after birth, plasma total corticosterone levels were similar in C-sectioned and vaginally born male pups. However, after birth by C-section there was dysregulation of total corticosterone, which appeared in two phases, i.e. first, a reduction in corticosterone starting very early, at 1 h after birth and also at 1 wk of age and second, an early rise of plasma corticosterone on d 14 away from the low values associated with the adrenal quiescent period in the first 1-2 wk in the rat.

Under all experimental birth conditions and at all time points examined, alterations in free, biologically active, corticosterone were reflected in changes in total corticosterone in both male and female animals, suggesting that either measure can be used as an adequate indicator of early developmental GC dynamics. It is noteworthy, however, that in both vaginally born control and experimental groups, the reduction in plasma CBG from birth to d 7 (e.g. CBG = 3.4% at d 7 compared with levels at birth, in vaginally born males) was greater than the reduction in plasma total corticosterone over the same time period (total corticosterone = 37.5% at d 7 compared with birth, in vaginally born males). This was reflected in a decreased percent binding of plasma corticosterone on d 7 compared with the value at birth and serves to dampen (but not to eliminate) the fall in free, biologically active, corticosterone on d 7.

Human neonates born by C-section without labor show reduced plasma cortisol in comparison with vaginally born humans(21–25). By contrast, in our rat model, C-sectioned neonates showed no difference (or increases) in plasma corticosterone immediately after birth, in comparison with vaginally born animals. There may be several reasons for these apparently discrepant observations in humans versus the rat. First, we attempted to perform C-section as close as possible to the time of natural birth to avoid the confound of prematurity. Thus some of the dams may have been in labor at the time of C-section in our study, and at least, in the case of humans, labor is known to be associated with increases in plasma corticosteroids(22, 24). Second, because corticosterone readily crosses the placenta in the rat(42), corticosteroids from both maternal and fetal sources may contribute to maintain corticosterone levels immediately after birth in both vaginally born and C-sectioned animals. In addition, rats born by C-section in our model could be somewhat stressed due to rapid maternal decapitation before delivery. This protocol was selected, however, because we wished to avoid the confound of anesthetic effects in these initial studies. In fact, our findings that plasma epinephrine levels are much lower immediately after C-section compared with vaginal birth (B. F. El-Khodor and P. Boksa, manuscript in preparation) suggests that maternal decapitation does not result in a measurable hormonal stress response in the pups. Third, our data from rats taken at 1 h after birth, rather than immediately after birth, may be more comparable to human data on GC levels at birth, because there is likely a variable lag period after birth before human blood samples are taken. Similar to data reported for human neonates delivered by C-section, rats born by C-section showed a marked reduction in corticosterone levels at 1 h after birth, in comparison with vaginally born controls. Thus although C-sectioned rats are born with plasma corticosterone levels comparable to those in vaginally born controls, the C-sectioned rat appears to lack a very early neonatal stimulus regulating corticosterone secretion or metabolism, resulting in reduced corticosterone levels by 1 h after birth.

In contrast to the C-section group, male pups born by C-section with 15 min of added anoxia had plasma corticosterone levels during the 1st wk that more closely resembled those of vaginally born animals. The anoxic group showed a less pronounced reduction in corticosterone at 1 h and normal corticosterone levels on d 1 and 7. It is possible that the stress of birth anoxia may activate systems that are responsible for the maintenance of plasma corticosterone levels during the 1st wk and that are inactive in the C-section alone group. However, anoxia did not influence the C-section-induced early rise in plasma corticosterone on d 14, because this phenomenon was also present in male pups born by C-section with added anoxia. Female pups born by C-section with added anoxia showed a significant reduction in corticosterone only on d 7. Surprisingly, anoxic female pups showed a significantly increased plasma corticosterone on d 1. These anoxic females killed on d 1 weighed considerably less than did females or males from all other groups. Because each litter contained males and females from experimental and control groups, it is possible that the anoxic females taken on d 1 may have been particularly stressed, due to their small size, resulting in higher plasma corticosterone. In females born by C-section with or without added anoxia, the early rise in plasma corticosterone on d 14 was not as pronounced as in males and, in fact, levels were not statistically different from those in vaginally born females.

It is unknown which features of the C-section birth and its sequelae are responsible for the observed alterations in plasma corticosterone during the first 2 wk of life. It is known, however, that vaginal birth and, to an even greater extent, fetal hypoxia during birth stimulate release of adrenal catecholamines in both humans and rats(43–45). Using the current rat model, we have recently demonstrated that plasma epinephrine levels at birth are lowest in male pups born by C-section, significantly higher in vaginally born animals, and much higher in pups born by C-section with 15 min of anoxia (B. F. El-Khodor and P. Boksa, manuscript in preparation). It is possible that circulating epinephrine or another factor activated by vaginal birth, but not by C-section, may interact to regulate the developmental profile of corticosteroid secretion during the 1st wk of life.

Animals delivered by C-section in the current study were born rapidly, began to breathe immediately and spontaneously, and exhibited color, muscle tone, respiratory rate, and activity levels similar to those in vaginally born animals. By contrast, pups born by C-section with 15 min of anoxia were dark in color, hypotonic and akinetic, and showed reduced respiratory rates during the first 15-20 min after birth. Males pups born by C-section showed a transient deficit in body weight during the 1st wk, compared with vaginally born males (Table 1). This suggests that poor growth might contribute to the altered levels of plasma corticosterone in C-sectioned males during this time. However, anoxic males showed weight reductions similar to those in C-sectioned males during the 1st wk, yet corticosterone levels were almost normal at this time in the anoxic group. Similarly, in female animals, reductions in body weight did not appear to readily correlate with changes in plasma corticosterone. Thus factors other than body weight must also contribute to development of the plasma corticosterone profile during the first weeks of life.

What might be the short- or long-term consequences of alterations in plasma corticosterone during the first 2 wk of life? First, it has been reported that the frequency of respiratory distress syndrome type II, a form of transient respiratory morbidity that usually improves by 24-48 h of age, is greatest for term human neonates born by C-section without labor, of intermediate frequency after C-section birth with labor, and of lowest frequency after vaginal birth(26). Because lack of the corticosteroids may retard appropriate lung maturation, it is possible that reductions in plasma corticosterone persisting through the 1st wk of life could contribute to early respiratory morbidity. Second, it has been suggested that the adrenal quiescent phase from d 3 to 14 in the rat may serve to suppress the catabolic effects of corticosterone at a time of much anabolic activity in the developing animal(16). Furthermore, there is strong evidence that the rise in GCs at the beginning of the third postnatal week in the rat plays an important role in the coordination of maturational events in various organs systems, such as the liver and the gastrointestinal system(11). At present, we lack information as to whether the precise timing of the adrenal quiescent phase and the subsequent rise of plasma corticosterone may differentially affect the development of various body tissues. If precise timing is critical, an early rise in plasma corticosterone in the 2nd wk may have developmental repercussions. We have previously shown that rats born by C-section when adults behave normally on a gross behavioral level and show no deficits in tests of spatial learning and sensorimotor performance(34). However, in adult animals that had been born by C-section, hippocampal and hypothalamic type I corticosteroid receptors show reduced affinity for corticosterone, and the plasma corticosterone response to acute restraint stress is blunted(46). Additionally, we have recently demonstrated alteration in steady state levels of dopamine in several brain regions from adult rats that had been born by C-section and enhanced release of dopamine from the nucleus accumbens in response to repeated stress in both C-sectioned and anoxic animals (B. F. El-Khodor and P. Boksa, manuscript in preparation)(47). Thus C-section birth appears to result in long-term alterations in components of the stress response in this model. Whether these more long-term changes may be influenced by the early developmental alterations in plasma corticosterone levels remains to be determined.

In conclusion, although basal plasma corticosterone levels measured at birth or at 5 wk of age are unaffected by route of delivery in the rat, C-section birth, with or without an added period of acute anoxia (in comparison with vaginal birth) does have significant effects on plasma corticosterone during the intervening early weeks of development, a time period thought to be particularly critical for effects of corticosteroids on developing tissues.