INTRODUCTION

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TRANSITION FROM FETAL TO NEWBORN life is accompanied by a marked rise in circulating norepinephrine (NE) concentrations in both animals (12, 23, 24) and infants (11, 14, 15). However, there is no significant change in arterial blood pressure associated with this dramatic rise in circulating NE during transition (23, 24, 30). The mechanism for this fetal/neonatal regulation of arterial pressure in response to circulating NE is not known. It has been demonstrated that the central nervous system, particularly the medulla, is responsible for regulation of normal cardiovascular function in fetal lambs (6). The nucleus tractus solitarius (NTS) is the principal sensory nucleus for central regulation of cardiovascular function (1, 32).

Recent studies (8, 27) have shown that nitric oxide (NO) in the NTS plays an important role in the central inhibition of sympathetic tone and thus decreases arterial blood pressure. Microinjections of nitroglycerin (NTG), an NO donor, into the NTS produce hypotension and bradycardia (19, 20), and L-arginine-derived NO directly affects the activity of central autonomic neurons in the NTS (16). Neuronal NO synthase (nNOS), which catalyses the transformation of arginine to NO, is functionally regulated and induced in neurons in the brain (5, 29). We have recently observed that nNOS immunoreactivity and NADPH diaphorase (NADPHd) reactivity are predominately enhanced in the medial NTS (mNTS) of the newborn compared with the fetus (18). Coexistence of NADPHd/nNOS and tyrosine hydroxylase (TH) in the brain nuclei of adult rats suggests that noradrenergic neurons are capable of generating NO for regulation of noradrenergic activity within the brain (25, 31).

The purpose of the present study was to determine the influence of the transition at birth on the coexistence of nNOS within noradrenergic A1 neurons and to examine whether transition enhanced nNOS expression in noradrenergic neurons in the brain stem nuclei, particularly in the NTS, in the neonate 4 h after birth vs. the term fetus. NO in the fetal medulla on central cardiovascular functions were investigated by fourth cerebral ventricle (4V) administration of NTG and an inhibitor of NOS in chronically instrumented fetal sheep in vivo.

	MATERIALS AND METHODS

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Animals. Experiments were performed by using time-dated gestational ewes with fetuses (Western, mixed-breed sheep from Nebeker Ranch, Palmdale, CA). The animals were maintained on a 12:12-h light-dark cycle in individual steel study cages. The protocol was approved by the Harbor-University of California at Los Angeles Animal Use Committee and was in accord with Association for Assessment and Accreditation of Laboratory Animal Care and National Institutes of Health guidelines. Food (alfalfa pellets) and water were available ad libitum except for the withholding of food 24 h before surgery.

Chronically instrumented fetal preparation. Six time-dated pregnant ewes with singleton fetuses (134-136 days gestation) were studied by using chronically instrumented preparation as previously described (9, 10). Surgical anesthesia of the ewe was induced with ketamine hydrochloride (20 mg/kg im). General anesthesia was maintained with isoflurane (3-4%) and oxygen (1-2 l/min) via a mask until placement of a cuffed endotracheal tube. After tracheal cannulation, the animals were ventilated at ~20 breaths/min with a tidal volume of 400-550 ml throughout the experiment. Isoflurane was titrated to 1-2%. The animals were placed on a water pump heating pad to maintain body temperature at 37°C. The maternal abdomen was opened via a midline incision, and the fetal head was extracted from the uterus. After placement of a maternal femoral catheter, arterial blood pressure, heart rate, and temperature were monitored continuously. A 4V catheter was placed in the vicinity of the area postrema (for administration close to the region of the NTS). An intrauterine catheter was inserted for amniotic fluid pressure measurement. Maternal femoral catheters were also placed, and all catheters were passed through a pouch on the side of the ewe. Uterine and maternal incisions were repaired and animals were allowed >3 days for postoperative recovery, which included catheter maintenance and antibiotic administration.

4V injection and histological examination. After postoperative recovery, fetal arterial blood pressure (corrected for amniotic cavity pressure) and heart rate were continuously monitored before and after 4V injection. An equilibration period was included for preparation of the animals for the experiments, and arterial blood pressure and heart rate were allowed to be stable for at least 30 min. Compounds were dissolved in artificial cerebral spinal fluid (aCSF, pH adjusted to 7.4) and were given in a volume of 200 µl over a period of 5 min (22, 23). NTG, an NO donor (1.2 nmol, 200 µl) or the same amount of aCSF was injected into the 4V. After a 2-h observation and equilibration, N^G-nitro-L-arginine methyl ester (L-NAME; an inhibitor of NOS, 10 nmol, 200 µl) was injected into the 4V, and fetuses were monitored for a final 2 h. We previously demonstrated that intravenous injection of NTG and L-NAME with the same doses did not cause changes in arterial blood pressure and heart rate in the ovine fetus (unpublished observations). 4V injections of the same amount of aCSF served as control. Each animal received two injections separated in time by 2 h. Throughout the experiments, fetal and maternal arterial blood pressure, heart rate, and amniotic fluid pressure were monitored continuously by using World Precision Instruments (Sarasota, FL) signal conditioners and transducers. One-minute segments of fetal arterial blood pressure were analyzed at 1 and 2 h as the control period and at 5, 10, 15, 20, and 30 min after injection of each compound. Effects of the administered compound are expressed as maximum changes obtained at 5-20 min after the injection compared with the baseline measurements established at 1 and 2 h before the injection.

Surgical preparation for histochemistry studies. Five time-dated gestational (147-148 days) ewes with twin fetuses were studied as previously described (18). Surgical anesthesia in ewes was induced with ketamine hydrochloride (20 mg/kg im) and maintained with subarachnoid and epidural injection of 2% lidocaine (5 ml) plus 0.5% bupivacaine (5 ml). The maternal abdomens were opened via a midline incision, a small hysterotomy was made, and the fetal head was extracted from the uterus. Polyethylene catheters were immediately placed in the fetal carotid artery for recording of arterial blood pressure. Arterial blood pressure was measured by means of a World Precision Instruments signal conditioner with transducers. After arterial blood was taken for measurement of NE concentration, a cannula was implanted into the carotid artery for immediate perfusion, and both sides of the jugular vein were cut in the fetal lambs under anesthesia with ketamine (20 mg/kg im) and acepromazine (10 mg/kg im). Perfusion was performed by using 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer for 45 min (13, 18).

NADPHd staining combined with TH immunohistochemistry. NADPHd staining was performed in the brain stem sections by using previously described techniques (18). Briefly, free tissue sections of the brain stem were incubated in 0.1 M Tris · HCl (pH 8.0)-0.3% Triton X-100 containing 1.0 mM reduced -NADPH and 0.2 mM nitro blue tetrazolium at 37°C for 90-120 min (7, 17, 33). The reaction was stopped by washing the sections with PBS (pH 7.4). The sections were then incubated in PBS containing polyclonal TH antibody (1:800 dilution) and 3% normal goat serum overnight at 4°C. The antigen-antibody complex was visualized by using the diaminobenzidine method as described by the manufacturer (Zymed Laboratories, South San Francisco, CA) by using streptavidin-biotin amplification (18, 25). The sections were mounted onto subbed slides and examined under a light microscope.

Cell counts and data analysis. Brain stem sections were examined under a light microscope. The TH-immunoreactive cells showed the characteristic brown staining of oxidized diaminobenzidine as the TH label. NADPHd reactivity was visualized as a vibrant blue color within perikarya, dendrites, and axons. These two distinguishable colors were used to identify the presence of TH immunoreactivity, NADPHd reactivity, and the codistribution of TH label and NADPHd staining in the cells (25). Micrographs were quantified by using a microscope with a reticule grid to measure the number of positive cells containing color staining in each nucleus. The number of positive cells in a section area (200 × 200 µm) was counted in five separate medulla sections containing both the rostral and caudal levels of the mNTS per animal, and an average number of positive neurons for each animal was obtained (17, 18). Changes in TH immunoreactivity, NADPHd reactivity, and codistribution in each brain stem nucleus were determined by expressing the number of positive cells per section area (200 × 200 µm). The quantitation was performed in a blinded fashion and determined in the mNTS, the commissural NTS, the rostral ventral medulla (RVM), and the gracile nucleus. Quantitation was determined in a blinded fashion for sections of medulla of all subjects. The levels of TH immunoreactivity, NADPHd reactivity, and double-labeled cells with NADPHd staining and TH immunoreactivity in each brain stem nucleus were compared in the fetus versus newborn.

	RESULTS

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Fetal cardiovascular responses to 4V administration of NTG and an inhibitor of NOS. To determine the effects of NO in the medulla on fetal cardiovascular regulation, fetal arterial blood pressure and heart rate were observed after 4V administration of an NO donor and an inhibitor of NOS in chronically instrumented term fetal sheep (n = 6). Figure 1 shows fetal MAP and heart rate before and after 4V administration of NTG and L-NAME. Fetal MAP was significantly decreased by 4V administration of NTG (1.2 nmol) and fetal heart rate was moderately reduced by the treatment (Fig. 1). 4V administration of L-NAME (10 nmol) caused a significant increase in fetal MAP but did not alter fetal heart rate (Fig. 1). Histological examination showed that the major blue dye was distributed around the membrane of the medulla with mainly staining in the region of the NTS and area postrema, and light dye staining partly existed in the cerebrum and cervical part of the spinal cord after 4V injections.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of mean arterial blood pressure (MAP) and heart rate responses to fourth ventricular (4V) administration of nitroglycerin (NTG; 1.2 nmol) and NG-nitro-L-arginine methyl ester (L-NAME; 10 nmol) in chronically instrumented near-term fetal sheep. A: fetal MAP is reduced by NTG and increased by L-NAME injected into the 4V. B: fetal heart rate is moderately decreased by 4V injection of NTG but is not altered by administration of L-NAME. Each bar represents the mean ± SE. *P < 0.05 (ANOVA) compared with controls (n = 6).

Influence of fetal-to-neonatal transition on coexistence of TH and NADPHd in the brain stem. Double labels with NADPHd staining and TH immunohistochemistry were carried out in five fetuses compared with five newborn lambs. Figures 2 and 3 show that the mNTS of fetal and newborn lambs exhibited many densely stained NADPHd medium-sized neurons and nerve fiber networks. As shown in Fig. 3, the mNTS exhibited more neurons containing densely stained NADPHd in a newborn compared with a fetal lamb. The number of NADPHd-positive cells per section area (200 × 200 µm) of the mNTS was markedly increased in the newborn lambs compared with the fetuses (Fig. 4, P < 0.05). These findings are consistent with the results of previous studies of the mNTS in fetal and newborn lambs (18). The number of NADPHd-positive cells in the mNTS in fetal and newborn lambs are similar to the previous results obtained with antibody raised against nNOS (18).

View larger version (112K): [in this window] [in a new window]   Fig. 2.   Low-magnification micrographs from sections of the medulla show the structures and distributions of NADPH diaphorase (NADPHd) staining and tyrosine hydroxylase (TH) immunoreactivity in the medial nucleus tractus solitarius (mNTS). A: section of medulla from a term fetus (148 days). B: section of medulla from a newborn lamb (postnatal 4 h of age).

View larger version (109K): [in this window] [in a new window]   Fig. 3.   High-magnification micrographs show TH-immunoreactive neurons and NADPHd-containing neurons and axon terminals in the mNTS in a term fetus (A) compared with a newborn lamb at postnatal 4 h of age (B). Neurons and axon terminals containing NADPHd are visualized as blue staining (short arrows) and TH-positive cells (long arrows) are determined by the identified brown color. Double-labeled cells (arrowheads) with NADPHd staining and TH immunoreactivity are increased in the mNTS in a newborn lamb compared with a fetus.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Colocalization of NADPHd and TH immunoreactivity in the mNTS of a near-term fetus compared with newborn lambs at 4 h of postnatal age. NADPHd staining cells in the mNTS are markedly enhanced in newborn lambs compared with term fetuses. Each bar represents the mean ± SE; (n = 5). *P < 0.05 (ANOVA) compared with fetuses.

	DISCUSSION

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We examined the influence of the transition at birth on the coexistence of nNOS within noradrenergic A1 neurons in brain stem nuclei, particularly in the NTS, in term fetuses and cesarean section-delivered newborns. Effects of local NO on fetal arterial blood pressure and heart rate were investigated by the administration of NTG and an inhibitor of NOS into the 4V close to the NTS, an important autonomic integrative site in the brain stem, in chronically instrumented fetal sheep. The major new findings of this study are as follows: 1) NTG, an exogenous NO donor, produces a decrease in fetal arterial blood pressure associated with a moderately reduced heart rate in the 4V; 2) blocking NOS with L-NAME in the 4V increases fetal arterial blood pressure; and 3) TH-positive cells containing NADPHd reactivity are increased in the mNTS of newborn lambs compared with the fetuses. These findings show that the predominant effect of exogenous NO in the 4V is a decrease in fetal MAP, whereas blocking NOS with L-NAME had effects opposite to NTG on the fetal arterial blood pressure. The results are consistent with the results of previous studies of hypotensive responses to microinjection of an NO donor into the NTS observed in adult rats, but blunted heart rate responses to NO are observed in the ovine fetus compared with adult rats (19, 20, 27). Data suggest that administration of NO in the 4V, mainly distributed in the brain stem, particularly in the NTS, produces an inhibitory regulation of fetal arterial blood pressure but not heart rate. In addition, data presented support the previous results, which show that nNOS immunoreactivity and NADPHd reactivity are prominently enhanced in the mNTS during the transition at birth (18) and further suggest that fetal mNTS contains a group of neurons endowed with nNOS, which are the noradrenergic neurons of the A1 group. nNOS expression in the neurons is upregulated during the transition from fetal to newborn life.

NTS is the principal sensory nucleus for most cardiovascular and other visceral afferent fibers entering the central nervous system and plays an important role in the central regulation of arterial pressure and heart rate (1, 32). Physiological and anatomic studies have shown that the medial portion of the NTS is a region of dense cardiovascular innervation and mediates cardiovascular reflex responses (4, 21, 28). Anatomic and neurophysiological evidence suggest that neurons in the NTS-RVM-intermediolateral cell column constitute the central sympathetic regulation pathways. Synapses from the NTS project to the ventromedullary cardiovascular vasomotor center and then to the intermediolateral cell column, which consists of sympathetic preganglionic neurons of the spinal cord. NO, a reactive gas molecule, acts as a messenger in the brain, much like a neurotransmitter with a widespread signaling mechanism and function (22, 27). L-Arginine-derived NO affects the spontaneous discharge rate and influences the electrophysiological properties of neurons in the mNTS in the adult rat (16). Previous results in adult rats have demonstrated that mNTS microinjection of an NO donor produces hypotension and bradycardia, dependent on central noradrenergic mechanisms (19, 20). These studies, in chronically instrumented near-term fetal sheep, show that 4V administration of an inhibitor of NOS increases arterial blood pressure and injection of an NO donor into the 4V decreases arterial blood pressure. However, the fetal heart rate is less sensitive to NO compared with the bradycardic responses to NTG injected into the 4V in the adult rat (19, 20). Because our histological examination showed that the distribution of 4V-injected fluid exists mainly in the region of the brain stem particularly in the NTS, it is possible that the compounds might affect NTS neurons directly. Our findings would be consistent with this possibility, but we cannot exclude indirect influences of 4V NTG or L-NAME via effects on other brain nuclei, on other central nuclei projecting to NTS, or on the cerebral circulation. Moreover, the precise site of the neurons affected by the compounds applied is still unclear. A more sophisticated approach would be required to address this issue. Despite these limitations, our findings would be consistent with NO-mediated central regulation of arterial blood pressure in the NTS and demonstrate different responses to NO on heart rate between the ovine fetus and adult rats. The results suggest that the fetal medulla, particularly NTS neurons, is possibly affected by L-arginine-derived NO and plays a role in inhibitory regulation of arterial blood pressure.

It has been well documented that the transition from fetal to newborn life is accompanied by a marked rise in circulating NE concentrations (11, 12, 14, 15, 23, 24, 30). Previous studies (18) from this laboratory have demonstrated that MAP is similar between the fetus and the newborn even though the plasma NE concentration in newborns is more than twofold greater than fetal values. These results are consistent with other studies (23, 24, 30) that demonstrated that arterial blood pressure does not substantively change despite the postnatal catecholamine surge after birth. However, the mechanism for autonomic regulation of arterial pressure in responses to a dramatic rise in circulating catecholamines during transition is not known. Recent studies (34) have reported that the NE turnover rate in brain nuclei is associated with nNOS mRNA expression and NO content in chronic renal failure rats. In adult rats, coexistence of NADPHd/nNOS and TH in brain nuclei has been demonstrated and suggests that noradrenergic neurons are capable of generating NO for regulation of noradrenergic activity within the brain (25, 31). Our previous studies (18) have demonstrated that NADPHd reactivity is predominately enhanced in the mNTS accompanied by the same level of increased nNOS expression in the newborn compared with the fetus during birth transition. nNOS catalyses the transformation of arginine to NO, which plays an important role in the central inhibition of sympathetic tone and cardiovascular function in the NTS (8, 27). These findings, in fetal and newborn sheep, show that the mNTS contains a group of neurons endowed with NADPHd, which are the noradrenergic neurons of the A1 group, and that NADPHd/nNOS expression in the neurons is upregulated during transition at birth. It is well documented that noradrenergic transmission in the mNTS is important in central control of the circulation and that NE is active in decreasing sympathetic nerve activity and arterial blood pressure in this region (2, 3). These results support the previous rat studies (25, 31) and demonstrate that the rise in circulating catecholamines during transition induces upregulation of NADPHd/nNOS expression in mNTS noradrenergic neurons. It is possible that elevated noradrenergic activation in mNTS noradrenergic neurons induces upregulation of nNOS, and enhanced nNOS/NO in the neurons produces a central reduction of arterial blood pressure. Thus transition-induced nNOS-NO in the mNTS might play an inhibitory role in autonomic regulation of neonatal arterial blood pressure at birth.

With regard to NADPHd reactivity, it has been suggested that NADPHd histochemistry provides a specific histochemical marker for expression of nNOS, and NADPHd has been considered an nNOS. The colocalization of NADPHd and nNOS in the same neurons has been reported by several groups (7, 9, 13, 18, 25). However, reports indicate that some NADPHd activity might be unrelated to nNOS or noncolocalization. Investigators have obtained highly selective NADPHd staining, exhibiting perfect (>95%) colocalization with immunohistochemically detected nNOS by using vascular perfusion with paraformaldehyde followed by postfixation (13). Our previous studies, using methods described for perfusion and fixation, show that the numbers of nNOS-immunostaining cells were similar to those in NADPHd-positive cells in the brain stem nuclei of normal rats (17) and in the NTS of fetal and newborn sheep (18). The same methods have been performed in these studies. The present results further confirm that the numbers of nNOS immunostaining cells in the NTS, and the changes between fetus and newborn were similar to those in NADPHd-positive cells. Our findings support others who have identified NADPHd as a specific marker for expression of nNOS. Thus transition-induced increases in NADPHd-positive cells endowed with TH is a representation of upregulation of nNOS expression in the noradrenergic neurons of the A1 group in the mNTS.

In summary, arterial blood pressure is decreased by 4V administration of an NO donor and increased by an inhibitor of NOS in the near-term ovine fetus. TH-positive cells containing NADPHd reactivity are enhanced predominately in the mNTS at 4 h of neonatal age versus the term fetus. Results suggest that L-arginine-derived NO in the NTS produces a central reduction of fetal arterial blood pressure, and NADPHd/nNOS is upregulated in the noradrenergic neurons of the mNTS at birth. Enhanced nNOS-NO in the noradrenergic neurons of the A1 group may play an important role in neurocardiovascular regulation during the transition from fetal to newborn life.