American Journal of Respiratory and Critical Care Medicine

Rationale: Fluoxetine is a selective serotonin reuptake inhibitor antidepressant widely used by pregnant women. Epidemiological data suggest that fluoxetine exposure prenatally increases the prevalence of persistent pulmonary hypertension syndrome of the newborn. The mechanism responsible for this effect is unclear and paradoxical, considering the current evidence of a pulmonary hypertension protective fluoxetine effect in adult rodents.

Objectives: To evaluate the fluoxetine effect on fetal rat pulmonary vascular smooth muscle mechanical properties and cell proliferation rate.

Methods: Pregnant rats were treated with fluoxetine (10 mg/kg) from Day 11 through Day 21 of gestation.

Measurements and Main Results: Fetuses were delivered by cesarean section. As compared with controls, fluoxetine exposure resulted in fetal pulmonary hypertension as evidenced by an increase in the weight ratio of the right ventricle to the left ventricle plus septum (P = 0.02) and by an increase in pulmonary arterial medial thickness (P < 0.01). Postnatal mortality was increased among experimental animals, and arterial oxygen saturation was 96 ± 1% in 1-day-old control animals and significantly lower (P < 0.01) in fluoxetine-exposed pups (79 ± 2%). In vitro, fluoxetine induced pulmonary arterial muscle contraction in fetal, but not adult, animals (P < 0.01) and reduced serotonin-induced contraction at both ages (P < 0.01). After in utero exposure to a low fluoxetine concentration the pulmonary arterial smooth muscle cell proliferation rate was significantly increased in fetal, but not adult, cells (P < 0.01).

Conclusions: In contrast to the adult, fluoxetine exposure in utero induces pulmonary hypertension in the fetal rat as a result of a developmentally regulated increase in pulmonary vascular smooth muscle proliferation.

Scientific Knowledge on the Subject

Epidemiologic data suggest that antidepressants may increase the risk of neonatal pulmonary hypertension.

What This Study Adds to the Field

In contrast to the adult, fluoxetine exposure in utero induces pulmonary hypertension in the fetal rat as a result of a developmentally regulated increase in pulmonary vascular smooth muscle proliferation.

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), is often prescribed as an antidepressant to pregnant women (1). An association between the maternal use of SSRIs in the third trimester of pregnancy and persistent pulmonary hypertension of the newborn (PPHN) has been reported (2). Whether the association between exposure to this drug late in pregnancy and PPHN syndrome is a causal one is unclear and its mechanism remains elusive (3).

Paradoxically, fluoxetine has been shown to reverse experimentally induced pulmonary hypertension in adult rats (4). Sertraline, another SSRI available for therapeutic use, was also shown to protect adult rats from monocrotaline-induced pulmonary hypertension (5). In fact, a retrospective review of human subjects with pulmonary hypertension showed a tendency toward a lower risk of death among those receiving SSRI therapy (6).

Together, the available clinical and experimental published data raise an important question concerning whether the possible causal relationship between SSRI exposure and pulmonary hypertension is unique to the fetus. Therefore, the purpose of the present study was to expose rats to fluoxetine late in gestation and to assess its effect on the fetal pulmonary vasculature. We hypothesized that the effect of fluoxetine on the pulmonary vasculature is maturationally dependent.

Animals

Timed-pregnant female and nonpregnant adult Sprague-Dawley rats (Charles River, Montreal, PQ, Canada) weighing 250–300 g were used. All procedures were conducted according to criteria established by the Canadian Council on Animal Care and were approved by the Hospital for Sick Children Research Institute Animal Care Review Committee.

Chronic Fluoxetine Administration Studies

Pregnant rats (n = 11) were treated by gastric gavage with fluoxetine hydrochloride (Apo-fluoxetine, 20 mg/5 ml; Apotex Inc, Toronto, ON, Canada) at 10 mg/kg once per day from Days 11 through 21 of gestation (term, 22 d). This duration was chosen because it corresponds chronologically to 20–40 weeks of gestation in humans. It is also the time period identified in the epidemiological studies as associated with a higher prevalence of PPHN syndrome after in utero exposure (2). A higher than therapeutically employed dose of fluoxetine was used because, when compared with humans, the rat species has a higher rate of in vitro hepatic intrinsic clearance of fluoxetine (7). On the basis of the available pharmacokinetic data, the fluoxetine dose used in this study results in rat plasma drug concentrations similar to the human therapeutic range (8). Control rats (n = 9) received a similar volume of vehicle (manufactured by the Hospital for Sick Children Pharmacy, Toronto, ON, Canada). On Day 21 of gestation, the animals were anesthetized (pentobarbital sodium, 60 mg/kg, subcutaneous) and subjected to a cesarean section to rapidly deliver all fetuses.

The lung, heart, and placenta were obtained and together with fetal body weight were individually recorded. The placenta was gently dried by patting it once with absorbent paper, weighed, and snap-frozen for later processing. The lungs were dissected to remove the third-generation intrapulmonary arteries for myograph studies. The hearts were removed intact, placed in 4% buffered paraformaldehyde, and subsequently dissected to obtain the right ventricular wall and left ventricle plus septum weights. The right-to-left ventricular plus septum weight ratio (RV:LV plus septum) was used as a surrogate marker for right ventricular hypertrophy resulting from pulmonary hypertension, as commonly reported by others (9).

Lung Histology

Six randomly chosen fetuses from each group were prepared for lung histology. Immediately after delivery the fetal chest cavity was opened and the trachea was cannulated with a 24-gauge plastic cannula. The lungs were then inflated overnight with 4% paraformaldehyde at a constant pressure (20 cm H2O) and embedded in paraffin. A 4-μm section was cut and double-stained with hematoxylin–eosin and Masson trichrome to allow for clear demarcation of the internal and external elastic lamina, as well as the muscle layer. With the aid of a computerized image analyzer system (Openlab; Improvision, Coventry, UK) coupled with a fine-resolution microscope, the external and internal perimeters of each identifiable pulmonary artery were measured. Pulmonary arteries were distinguished from veins on the basis of the greater muscle and connective tissue observed in the former, and on the more peripheral position in the acinus of the latter. Only arteries that were cross-sectionally cut were measured. The internal elastic lamina was used to demarcate the internal arterial perimeter, whereas the external elastic lamina was chosen as the external arterial perimeter. The arterial lumen diameter was calculated as the internal perimeter divided by π. The arterial muscle layer was quantified by measuring the medial area (external – internal arterial area, in square millimeters), as we have previously described (10).

Pulmonary Arterial Smooth Muscle Cell Proliferation

Primary culture pulmonary arterial smooth muscle cells (PASMCs) were obtained from untreated adult and fetal Sprague-Dawley rats. After dissection and removal under sterile conditions, third-generation intrapulmonary arterial tissue was cut into small pieces and incubated in Dulbecco's modified Eagle's medium (DMEM) containing papain (0.5 mg/ml), albumin (1 mg/ml), and dithiothreitol (1 mg/ml), on ice for 15 minutes, at 37°C for an further 15 minutes, and then centrifuged at 1,000 rpm for 3 minutes The pellet was resuspended in DMEM with 10% fetal bovine serum and maintained in a humidified 95% air–5% CO2 incubator at 37°C. The cells were passaged by trypsinizing with 0.25% trypsin–ethylenediaminetetraacetic acid (GIBCO; Invitrogen, Burlington, ON, Canada) and used for experiments at passages 2–4. The specificity of the cultured cells was confirmed by immunostaining with a monoclonal antibody raised against smooth muscle α-actin (Roche Molecular Chemicals, Indianapolis, IN).

Cultured cells were first stained with the membrane-permeable nucleic acid stain 4′,6-diamidino-2-phenylindole (5 M; Invitrogen Molecular Probes, Eugene, OR) to estimate total cell number. Cells in medium supplemented with 15% fetal bovine serum were seeded on 24-well plates at a density of 5 × 104 cells per well and allowed to adhere. Cell number was determined with a Coulter particle counter (Beckman Coulter, Fullerton, CA). Growth was arrested in serum-free DMEM for 24 hours and the cells were incubated with either serotonin (10−4 M) or fluoxetine (10−8 to 10−4 M) for 24 hours and compared with nonsupplemented medium. Cell proliferation was measured by alamarBlue assay according to the manufacturer's instructions (BioSource Invitrogen, distributed by Medicorp, Inc., Montreal, PQ, Canada). Briefly, alamarBlue was added to the cell medium at a 1:10 (vol/vol) ratio and incubated for 12 hours at 37°C. Fluorescence was measured at excitation and emission wavelengths of 550 and 590 nm, respectively (SpectraMax Gemini EM; Molecular Devices, Sunnyvale, CA). The drug-induced proliferation rate was expressed as a percentage relative to untreated control cells.

Tissue Serotonin Concentration

A commercially available competitive serotonin ELISA kit (Labor Diagnostika Nord, Nordhorn, Germany) was used to determine the serotonin concentration in fetal lungs and placental tissue. Tissue was homogenized in the presence of Triton X-100, incubated for 30 minutes at 37°C, and centrifuged at 10,000 × g for 5 minutes, and the collected supernatant was used in the extraction procedure according to the manufacturer's recommendations. The serotonin tissue concentration was measured on the basis of the kit-generated standard linear curve fit.

Organ Bath Studies

Third- or fourth (adult)-generation left lung intralobar pulmonary arterial ring segments (average diameter, 80–100 mm; length, 2 mm) were dissected free and mounted in a wire myograph (Danish Myo Technology A/S, Aarhus, Denmark). Isometric changes were digitized and recorded online (MyoDaq; Danish Myo Technology A/S). Tissues were bathed in Krebs-Henseleit buffer (NaCl, 115 mM; NaHCO3, 25 mM; NaHPO4, 1.38 mM; KCl, 2.51 mM; MgSO4 · 7H2O, 2.46 mM; CaCl2, 1.91 mM; and dextrose, 5.56 mM) bubbled with air–6% CO2 and maintained at 37°C. After 1 hour of equilibration, the optimal tissue resting tension was determined by repeated stimulation with 128 mM KCl until maximal active tension was reached. All subsequent force measurements were obtained at optimal resting tension.

Pulmonary vascular muscle force generation was evaluated by stimulating with the thromboxane A2 mimetic U46619, fluoxetine, or serotonin. Contractile responses were normalized to the tissue cross-sectional area as follows: (width × diameter) × 2 and expressed as millinewtons per square millimeter (mN/mm2). The relaxant response to endothelium-dependent acetylcholine and endothelium-independent nitric oxide donor sodium nitroprusside was determined in fetal arteries after prestimulation with U46619 (10−6 M).

Postnatal Studies

Four fluoxetine-treated rats and one control rat were allowed to deliver spontaneously to evaluate the immediate postnatal course of the pups. From each fluoxetine-exposed litter a random sample of four animals was chosen for evaluation of vital signs and arterial oxygen saturation from birth through 3 days of life. The control pups were studied only on the first day of life.

To obtain the measurements the pups were briefly separated from the mother, placed on a warm pad maintained at 37°C, and evaluated while at rest. A pulse oximeter probe was fitted around the neck of each pup and arterial blood oxygen saturation was measured with a commercially available clinical pulse oximetry device (model N-30; Nellcor, Pleasanton, CA). Consistent and reproducible oxygen saturation measurements were obtained over a 3-minute period in all animals evaluated. The respiratory rate for each animal was obtained by visual inspection during the oxygen saturation measurement.

Drugs

Unless otherwise indicated, all drugs were obtained from Sigma-Aldrich (Oakville, ON, Canada).

Data Analysis

Data were evaluated by Student t test or two-way analysis of variance with multiple comparisons obtained by the Tukey–Kramer test when appropriate. Statistical significance was accepted at P < 0.05. All statistical analysis was performed with the Number Cruncher Statistical System (NCSS, Kaysville, UT). Data are presented as means ± SEM.

Body and Organ Weights

Fluoxetine exposure did not affect fetal weight gain. At delivery, fluoxetine-exposed (n = 88) fetal body weight was 3.8 ± 0.1 g and comparable to the 3.6 ± 0.0 g for the control group (n = 36). There was also no observed difference in tissue:body weight ratio between control and fluoxetine-exposed fetal lung (n = 17; 0.032 ± 0.001 vs. n = 8; 0.032 ± 0.001, respectively) and placenta (n = 29; 0.15 ± 0.01 vs. n = 27; 0.14 ± 0.01, respectively).

Pulmonary Hypertension

An 8 ± 2%, statistically significant (p = 0.02) increase in RV:LV+septum weight ratio was observed in fluoxetine-exposed fetuses (0.46 ± 0.01; n = 87) as compared with the control group (0.43 ± 0.01; n = 36), indicating right ventricular hypertrophy secondary to pulmonary hypertension. This was associated with a significant (P < 0.01) increase in fluoxetine-exposed fetal 50- to 200-μm vessel diameter pulmonary arterial medial thickness, when compared with the control group (Figure 1).

Fetal and Adult Pulmonary Arterial Smooth Muscle Contraction and Relaxation

The fetal pulmonary arterial muscle thromboxane A2 analog (U46619)–induced dose response was not altered in fluoxetine-exposed animals (Figure 2). Acetylcholine and sodium nitroprusside relaxation dose–response curves were obtained after a precontraction U46619-generated force of 2.2 ± 0.2 mN/mm2 in fluoxetine-exposed and 2.7 ± 0.2 mN/mm2 in control fetal pulmonary arteries (P = not significant). There was no significant difference in endothelial-dependent and -independent relaxation of fluoxetine-exposed vessels compared with control fetal pulmonary arteries (Figure 2).

When compared with their adult counterpart, control fetal pulmonary arteries showed less response to serotonin and significantly greater (P < 0.01) muscle contraction after fluoxetine stimulation (Figure 3). Fetal fluoxetine-exposed (n = 8) and control (n = 4) pulmonary arterial muscle fluoxetine-induced contraction were similar (Figure 3).

Preincubation (20 min) with fluoxetine significantly (P < 0.01) decreased the serotonin-induced force in adult and fetal control pulmonary arteries (Figure 4). Similarly, fluoxetine preincubation significantly (P < 0.01) reduced the U46619 (10−6 M)-induced fetal pulmonary arterial muscle force from 2.7 ± 0.1 to 1.6 ± 0.2 mN/mm2 (n = 4).

Pulmonary Arterial Smooth Muscle Proliferation

In response to serotonin stimulation (10−4 M) fetal PASMCs showed a significantly lower (P < 0.05) proliferation rate, as compared with adult cells (Figure 5). In contrast, when stimulated with various concentrations of fluoxetine, a significant (P < 0.01) difference between the fetal and adult PASMC proliferation rate was observed (Figure 6). At low concentrations (⩽10−7 M) fluoxetine significantly increased the fetal, but not the adult, PASMC proliferation rate (Figure 6). At higher concentrations (>10−7 M), fluoxetine decreased the proliferation rates of fetal and adult PASMCs. At all higher concentrations, however, the reduction in PASMC proliferation rate was significantly greater in adult, compared with fetal, cells.

Serotonin Tissue Concentration

To evaluate the effect of fluoxetine exposure on the lung and placenta, we measured the tissue serotonin concentration in treated and control fetuses. As compared with control animals, fluoxetine exposure did not alter lung serotonin content (32 ± 4 vs. 27 ± 3 ng/mg tissue weight), but significantly reduced (P < 0.01) placental tissue serotonin content (18 ± 3 vs. 7 ± 1 ng/mg tissue weight).

Postnatal Course of Fluoxetine-exposed Pups

Whereas no newborn deaths occurred in the control litter, the mortality rate for the 53 fluoxetine-exposed newborns was 15% over the first 72 hours of life. Among the survivors, there was a tendency (P = not significant) toward a higher respiratory rate (82 ± 6 breaths/min; n = 8) in fluoxetine-exposed pups compared with control pups (68 ± 5 breaths/min; n = 6) at 3 hours of age.

Figure 7 illustrates the daily arterial oxygen saturation values for the fluoxetine-exposed animals. Arterial oxygen saturation was 96 ± 1% (n = 6) in control animals and significantly lower (P < 0.01) in fluoxetine-exposed newborns (79 ± 2%; n = 8) at 3 hours of age. Arterial oxygen saturation in the experimental newborns progressively increased over time and was comparable to that of the control animals on the third day of life.

Major depressive disorders during pregnancy and in the immediate postnatal period are observed in 10% of women (11) and 2–3% of these are treated with SSRIs (12). In pregnant sheep (13), a transient decrease in uterine arterial blood flow and fetal blood oxygenation was previously reported following intravenous fluoxetine administration. Yet the SSRI exposure effect on the fetal pulmonary vasculature and hypertension has not been previously evaluated.

Fluoxetine is one of the most commonly used SSRIs for child-bearing women (14) and after its use during the third trimester of gestation a significant increase in transient mild neonatal clinical abnormalities was reported (15). These include transient respiratory distress, irritability, lethargy, feeding difficulty, and body temperature changes (16, 17).

After its administration to adult animals fluoxetine is maximally concentrated in the lung (18, 19). Yet, the mechanism by which SSRI exposure during late gestation might induce PPHN syndrome is unknown. In an attempt to explain their epidemiologic evidence, Chambers and coworkers (2) suggested that fluoxetine induces PPHN syndrome via a rise in serotonin levels, leading to pulmonary vasoconstriction and smooth muscle proliferative effects. Such speculation is justified on the basis of the evidence for overproduction of this compound in adult humans with idiopathic pulmonary hypertension (20).

This line of reasoning, however, disregards the accumulating evidence that fluoxetine is protective against monocrotaline- (4) and chronic hypoxia–induced (21) pulmonary hypertension in the adult rat and mice. In addition, the “protective” effect of fluoxetine in abrogating the pulmonary vascular remodeling process in pulmonary hypertension has been linked to its effect in decreasing pulmonary vascular smooth muscle proliferation (22). In this study we evaluated the fluoxetine effect on the fetal pulmonary vasculature, to reconcile the discrepancy between the newborn and adult data regarding its role in the pathogenesis of pulmonary hypertension.

The fluoxetine daily dose administered to pregnant rats was higher than therapeutically used in humans to overcome the higher rate of in vitro hepatic intrinsic clearance of fluoxetine in rats compared with humans (7). The fluoxetine dose administered to the rats in this study has been shown to result in plasma drug concentrations similar to the human therapeutic range (8).

In this study, we showed that chronic fluoxetine exposure starting in the latter part of gestation leads to fetal pulmonary hypertension in the rat. This is based on the increase in RV:LV+septum ratio, as a surrogate marker for a rise in pulmonary vascular resistance, and pulmonary arterial medial thickening indicative of vascular remodeling. In addition, fluoxetine in utero–exposed newborns showed lower arterial oxygen saturation immediately after birth and a higher mortality rate compared with control animals. As judged by the arterial oxygen saturation measurements, the pulmonary hypertension resolved within the first 3 days of life in the surviving pups.

To compare and evaluate the developmental pattern of response, we further measured the fluoxetine effect on fetal and adult pulmonary vascular smooth muscle cell proliferation rates. We documented that ex vivo, under culture conditions, primary fetal PASMCs proliferate at a faster rate than do adult cells when exposed to low concentrations of fluoxetine (23). Available data on the fluoxetine volume of distribution and pharmacokinetics during pregnancy strongly suggest that the fetal rat lung drug levels in this study were likely in the lower range tested in vitro for the PASMC proliferation studies (7, 8, 23). In addition, although at higher than 10−7 M fluoxetine decreased the PASMC proliferation rate, the reduction was consistently less in fetal, as compared with adult, cells at all concentrations tested.

The possibility that the mechanism responsible for the in utero fluoxetine-induced PPHN involves a direct inhibitory effect of the drug on pulmonary vascular nitric oxide production has been suggested (2). The data from this study do not support such speculation. The near-resistance intrapulmonary arteries of the fluoxetine-exposed fetuses, in this study, showed a similar relaxant response to endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitroprusside) relaxation. This suggests preserved endothelial nitric oxide synthase activity and intact nitric oxide–mediated fetal pulmonary arterial muscle relaxation following fluoxetine exposure.

It is equally unlikely that the fluoxetine-induced pulmonary hypertension is related to serotonin-mediated pulmonary vasoconstriction, as speculated by Chambers and coworkers (2). The lung tissue serotonin level in this study was similar and the placental tissue concentrations were lower in the fluoxetine-exposed fetuses, as compared with the control animals. This lower serotonin level has also been reported in the cord blood of neonates who have been exposed in utero to fluoxetine (24).

As previously shown by others in the adult rat (25) and confirmed in the present study, fluoxetine reduces serotonin-induced pulmonary arterial muscle contraction via drug-related serotonin receptor blockade. This is further supported by the similar fluoxetine reducing effect on thromboxane A2 analog–induced contraction of fetal pulmonary arteries observed in this study. If anything, such a fluoxetine effect would have “protected” the exposed fetuses from developing pulmonary hypertension. Thus, our data make it unlikely that fluoxetine exposure in utero results in the PPHN syndrome on the basis of enhanced serotonin-mediated pulmonary vasoconstriction.

In contrast to the adult, where no response was seen, the fetal pulmonary arteries in this study exhibited a contractile response to fluoxetine that was similar for control and in utero fluoxetine–exposed animals. This lack of force difference to fluoxetine stimulation among the two groups of fetal arteries (Figure 3) is not surprising given their similar response to the thromboxane A2 analog (Figure 2). In the adult rat, intravenous fluoxetine administration caused a small increase in pulmonary arterial pressure and further depolarized pulmonary arterial smooth muscle cells via membrane K+ current inhibition (26). Whether fetal pulmonary arterial smooth muscle is more sensitive to K+ current inhibition as compared with the adult cells is presently unknown. We have previously shown that in rats the inward rectifier K+ blocker BaCl2 induced phasic contractions in nonstimulated fetal, but not adult, bronchial muscle (27). Thus the fetus-specific effect of fluoxetine on the pulmonary vasculature resistance could have played a small part in the drug-induced developmental differences in pulmonary vascular tone.

Fluoxetine is known to inhibit the serotonin transporter (5-hydroxytryptamine transporter [5-HTT]) (21). This transporter is responsible for the smooth muscle proliferative effect of serotonin (28). In 5-HTT overexpressor mice a significant increase in right ventricular pressure has been noted and an enhanced response to hypoxia was documented (29). Developmentally, little is know about 5-HTT expression in the lung. In brain tissue 5-HTT expression increases during gestation and the immediate neonatal period (30, 31). Therefore, given the likely lower 5-HTT expression in utero and the fluoxetine inhibitory effect of this transporter it is unlikely that the fetal pulmonary hypertension documented in this study results from increased 5-HTT activity. In summary, in contrast to its protective effect in the adult, fluoxetine induces fetal pulmonary hypertension. The mechanism responsible for this developmentally unique effect of the drug appears to involve increased fetal PASMC proliferation and consequent pulmonary vascular remodeling.

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Correspondence and requests for reprints should be addressed to Jaques Belik, M.D., Division of Neonatology, University of Toronto Hospital for Sick Children, Room 3886, 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail:

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