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. 2016 Jun 21;153(1):103–111. doi: 10.1093/toxsci/kfw108

From the Cover: Prenatal Nicotinic Exposure Attenuates Respiratory Chemoreflexes Associated With Downregulation of Tyrosine Hydroxylase and Neurokinin 1 Receptor in Rat Pup Carotid Body

Lei Zhao *, Jianguo Zhuang *, Xiuping Gao *, Chunyan Ye *, Lu-Yuan Lee #, Fadi Xu *,1
PMCID: PMC5841596  PMID: 27329243

Abstract

Maternal cigarette smoke is the major risk of sudden infant death syndrome (SIDS). A depressed ventilatory response to hypoxia (HVR) and hypercapnia (HCVR) is thought to be responsible for the pathogenesis of SIDS and the carotid body is critically involved in these responses. We have recently reported that prenatal nicotinic exposure (PNE) over the full gestation induces depressed HVR in rat pups. Here, we asked whether PNE (1) depressed not only HVR but also HCVR that were dependent on the carotid body, (2) affected some important receptors and neurochemicals expressed in the carotid body, such as tyrosine hydroxylase (TH), neurokinin-1 receptor (NK1R), and α7 nicotinic acetylcholine receptor (α7nAChR), and (3) blunted the ventilatory responses to activation of these receptors. To this end, HVR and HCVR in Ctrl and PNE pups were measured with plethysmography before and after carotid body ablation (Series I), mRNA expression and/or immunoreactivity (IR) of TH, NK1R, and α7nAChR in the carotid body were examined by RT-PCR and immunohistochemistry (Series II), and the ventilatory responses were tested before and after intracarotid injection of substance P (NK1R agonist) and AR-R17779 (α7nAChR agonist) (Series III). Our results showed that PNE (1) significantly depressed both HVR and HCVR and these depressions were abolished by carotid body ablation, (2) reduced the relative population of glomus cells, mRNA NK1R, and α7nAChR and IR of NK1R and TH in the carotid body, and (3) decreased ventilatory responses to intracarotid injection of substance P or AR-R17779. These results suggest that PNE acting via the carotid body could strikingly blunt HVR and HCVR, likely through downregulating TH and NK1R.

Keywords: SIDS, maternal cigarette smoke, carotid body ablation, glomus cells, dopamine D2 receptor, ventilatory response to hypoxia and hypercapnia.

Maternal cigarette smoke during pregnancy is one of the highest risk factors for sudden infant death syndrome (SIDS) and nicotine is thought to be a major player (Duncan et al., 2008; Slotkin and Seidler, 2011). Some victims of SIDS and infants at high risk of SIDS reportedly present depressed ventilatory response to hypoxia (HVR) and hypercapnia (HCVR) (Gaultier, 2000; Kahn et al., 2003; Slotkin et al., 1995; Valdes-Dapena, 1980) that is assumed to be involved in SIDS pathogenesis (Harris and St-John, 2005; Ueda et al., 1999). Prenatal nicotinic exposure (PNE) has been utilized to develop SIDS animal models. However, the traditional PNE that is usually administrated over the last two-thirds of the 21-day gestation in rats induces a mildly blunted (Eugenin et al., 2008) or unchanged HVR (Bamford and Carroll, 1999; Robinson et al., 2002) in neonates. The same controversy was also observed in HCVR (Bamford and Carroll, 1999; Eugenin et al., 2008; Kahn et al., 2003). We recently developed a “full term” PNE over the full gestation and postnatal period up to postnatal day 12–14 (P12–14) in which HVR depression was obvious (38%↓) (Zhuang et al., 2014). However, the effect of “full term” PNE on HCVR has not yet been investigated, despite the important function of HCVR in regulating the normal physiological conditions in neonates.

The initial HVR (within 1–2 min of hypoxia) is mediated by peripheral arterial chemoreceptors, predominantly by the carotid body (Lopez-Barneo et al., 2004; Prabhakar, 2000), whereas the following phase of HVR is the sum of peripheral stimulation of the carotid body (Ang et al., 1992; Prabhakar, 2000; Vovk et al., 2004) and a central depressant effect (van Beek et al., 1984; Yelmen, 2004). With respect to HCVR, though it is dominantly mediated by central CO2-chemoreception, its full expression also requires the carotid body (Carroll, 2003; Gauda and Lawson, 2000). An abnormality of the carotid body in SIDS victims has been proposed due to a decrease in the number of glomus (Cole et al., 1979). On the other hand, a recent study showed that in preterm infants, prenatal smoking exposure changed chemoreceptor tonic activity in a sleep-state dependent manner, pointing to a central involvement (Stephan-Blanchard et al., 2010). To date, it remains unclear to what extent the depressed HVR and HCVR triggered by PNE depends on the integrity of the carotid body.

In our pilot study, the depressed HVR and HCVR were no longer observed in the PNE pups with carotid body ablation, which suggested an adverse impact of PNE on the carotid body. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamine and its biosynthesis is reportedly increased when the carotid body is activated (Czyzyk-Krzeska et al., 1992; Gonzalez et al., 1981). The immunoreactivity (IR) of TH in the carotid body has been used to reflect the relative population of glomus cells and the carotid body sensitivity (Chai et al., 2011; Yamaguchi et al., 2003). In addition, multiple receptors, such as neurokinin-1 receptor (NK1R), α7 nicotinic acetylcholine receptor (α7nAChR), and dopamine 2 receptor (DA2R), are located at glomus cells and/or the terminals of the carotid sinus nerve (Nurse, 2010; Nunes et al., 2014; Stephan-Blanchard et al., 2013). Intracarotid injection of substance P via action on NK1R (Cragg et al., 1994) or nicotine/acetylcholine via α7nAChR (Nurse, 2010; Shirahata et al., 1998) could stimulate the carotid body and increase ventilation. Consistent with these findings, blockade of the carotid body NK1R attenuated HVR in rats (Cragg et al., 1994; De Sanctis et al., 1994). In addition, intracarotid injection of dopamine decreased ventilation (Monteiro et al., 2009) by binding to DA2R (Nurse, 2010; Prabhakar, 2000). These lines of information allow us to ask if PNE is capable of altering TH and these receptors in the carotid body and, if so, whether these alterations would change the ventilatory responses to activation of these receptors.

MATERIALS AND METHODS

Ten male and 34 female pathogen-free Sprague Dawley rats (250–350 g) were purchased from Charles River Laboratories, Inc. (Wilmington, Massachusetts) and quarantined for 2 weeks before the experiments. The experimental protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, USA.

Pretreatment with PNE

The females were randomly designated to receive vehicle (n = 17) and nicotine (n = 17), respectively. Briefly (Zhuang et al., 2014), an osmotic minipump (0.25 μl/h for 28 days, Alza Corp., Palo Alto, CA) was subcutaneously implanted in the females to deliver vehicle or nicotine tartrate (6 mg/kg/day) that produces nicotine blood levels approximately equivalent to those that occur in moderate to heavy smokers (Slotkin et al., 1997). Ten days after the implantation, each female rat was placed in a breeding cage with a male rat for up to 4 days. Those with vaginal plugs were considered pregnant and separated from the male. On the seventh day of gestation, the minipump was replaced with a new one filled accordingly with vehicle or nicotine. Only 1–2 male pups from each litter with similar overall litter size was/were selected for a given experimental in each study series to avoid the possible genetic interference with the results. Males at P12–14 were chosen in this study because males are much more vulnerable than females in human SIDS (Adams et al., 1998) and pups’ nerve development at this period is equivalent to newborn infants at 2–4 months (Ballanyi, 2004). Pups from vehicle- and nicotine-treated dams were grouped as Ctrl and PNE and randomly assigned to the following studies.

Experimental protocols

Study Series I was designed to examine if PNE was able to change HVR and HCVR dependent on the carotid body. After habituation, the pups at P12 (n = 10 for Ctrl and PNE, respectively) were placed in a plethysmograph chamber (Buxco Electronics Inc., Wilmington, NC) continuously flushed with normoxic (21% O2 and 79% N2) gas mixtures at 0.5 l/min (Zhuang et al., 2014). After stabilization, they were randomly exposed to hypoxia (5% O2, balanced with N2) and hypercapnia (7% CO2, 40% O2, balanced with N2) for 5 min. Subsequently, of these, 5 Ctrl and 8 PNE pups were anesthetized by isoflurane with retromandibular neck incisions made under sterile condition. The carotid bifurcation and the carotid body were bilaterally and carefully exposed from a ventral approach and the carotid body was gently removed with Surgicel to block the bleeding, if it occurred, as previously reported (Zhang et al., 2011). Immediately after carotid body ablation, each animal was exposed to the same hypoxia during anesthesia, and the efficacy of the ablation was validated by the lack of HVR. Two days after recovery, the animals were exposed to the same hypoxic and hypercapnic challenges in the chamber.

Study Series II was conducted to test whether PNE affected the carotid body by alteration of (1) the density of TH-IR, (2) the relative population of glomus cells, (3) gene levels of NK1R, α7nAChR, and DA2R, and (4) NK1R-IR and α7nAChR-IR. We did not test DA2R-IR because of the lack of PNE effect on mRNA DA2R in our pilot study. The pups (n = 20 and 19 for Ctrl and PNE) were euthanized. The carotid body was harvested and the fresh tissues were prepared for RT-PCR to detect the mRNA levels in 7 Ctrl and 7 PNE pups. In remaining pups (13 for Ctrl and 12 for PNE), the carotid body was fixed and prepared for IR. TH-IR coupled with NK1R-IR (n = 6 for Ctrl and PNE pups) or α7nAChR-IR (n = 7 and 6 for Ctrl and PNE pups, respectively) were performed.

Study Series III was performed to determine if manipulation of NK1R, α7nAChR, and DA2R of the carotid body would affect ventilatory responses differently in Ctrl and PNE pups. The pups were anesthetized with urethane (1200 mg/kg, intraperitoneally). As needed, supplemental urethane (300 mg/kg, intraperitoneally) was administered to completely eliminate eye-blink and limb withdrawal reflex throughout the experiment. Under adequate anesthesia, a catheter (PE 50-gauge tubing) was cannulated into the right common carotid artery until its tip was placed ∼3 mM caudal to the carotid bifurcation as reported before (Wang et al., 2006) for intracarotid injection. Because PNE downregulated the receptors of NK1R and α7nAChR expression in the carotid body, the ventilatory responses to intracarotid injection of substance P (3 mg/kg; NK1R agonist, n = 7/group) or AR-R17779 (30 mg/kg, α7nAChR agonist, n = 5/group) or their vehicles were recorded. An interval of 30 min was allowed between the 2 injections. Though DA2R (gene level) of the carotid body lacks a remarkable change in this study, its function and/or ligand endogenously released is possibly altered by PNE to contribute to the PNE-induced HVR depression. This assumption is supported by our previous study in which PNE failed to alter TRPV1 gene (transient receptor potential cation channel subfamily V member 1) in pulmonary C fibers, but dramatically prolonged apneic response to right atrial injection of capsaicin (an agonist of TRPV1) (Zhao et al., 2016). Therefore, HVR to 2 min of 10% O2 challenge was performed before and 10 min after intracarotid injection of peripheral DA2R antagonist (domperidone, DOM, 1 mg/kg) in anesthetized and spontaneously breathing Ctrl and PNE pups (n = 6/group). The doses of these agents utilized in this study were the same as previously used in intracarotid injections in rats without other side effects (Clark et al., 2013; Kotani et al., 1981; Maier et al., 2011).

Ventilatory measurements in anesthetized pups

As previously reported (Wang et al., 2006), under adequate anesthesia the trachea was cannulated and connected to a pneumotachograph (Frank’s Mfg. Co., Albuquerque, NM) to record airflow. The animals were exposed to a gas mixture of 40% oxygen in nitrogen (as a baseline), hypoxia, or hypercapnia. The core temperature of the animal was monitored with a rectal probe and maintained at ∼36 °C with a heating pad and radiant heat. A carbon dioxide analyzer (MicroCapStar end-tidal carbon dioxide analyzer, Model 15-10000; CWE, Inc. Ardmore, PA) was connected to a side-port of the tracheal cannula to monitor the end-tidal pressure of carbon dioxide (PETCO2).

mRNA analysis

The carotid body was collected and stored in −80 °C immediately. Total mRNA in the carotid body was isolated using RNeasy Mini Kit (Qiagen 74104, German), and cDNA was synthesized by reverse transcription using Sensiscript RT Kit (Qiagen 205211, German). TaqMan real-time PCR was conducted on ABI PRISM 7900 HT system (Applied Biosystems) to measure NK1R (NM_012667.2), α7nAChR (NM_012832.3, XM_008759497.1, XM_008759498.1), and DA2R (NM_012547.1, XM_006242979.2, XM_008766190.1, XM_008766191.1) in cDNA using the ΔΔCT method. Glyceraldehyde 3-phosphate dehydrogenase (NM_017008.3) was used as the endogenous control. Reaction conditions were carried out as follows: 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 72 °C for 30 s.

Immunohistochemical labeling

After euthanasia, intracardiac perfusion of 0.1 M phosphate-buffered saline (PBS, pH 7.4) and then 4% paraformaldehyde in PBS were conducted. The carotid body was bilaterally removed and embedded into Cryomold molds prefilled with optimal cutting temperature compound, and then frozen rapidly to about –23 °C. The frozen block was transected with the thickness of each slice at 8 μM. The samples were permeabilized and non-specifically blocked with the solution containing 1% bovine serum albumin and 0.3% Triton X-100 in PBS at room temperature for 2 h. Subsequently, they were incubated for 48 h at 4ºC with primary antibodies (Rabbit anti-TH pAb, Millipore, 1:500; Guinea pig anti-NK1R pAb, Millipore, 1:500; goat anti-α7nAChR pAb, Novus, 1:400), and visualized using Alexa Fluoro 488/594 (1: 400, Life Technologies) at room temperature for 2 h. The omission of primary antibodies was included as negative control. After immunohistochemistry, no more than 3 sections with the largest cross-section area of each carotid body were collected for imaging and the data averaged. The images were acquired using a 10× water immersion objective from the luminal surface with a digital camera (AxioCam HRm, Zeiss, Germany) connected to an epifluorescence microscope (Axioplan 2 FS, Zeiss, Germany). To detect the relative population of glomus cells (Yamaguchi et al., 2003), the ratio (%) of total areas with TH-IR (please see Figure 3) over the cross-section area of the carotid body at a given slice was calculated.

FIG. 3.

FIG. 3.

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Comparison of tyrosine hydroxylase (TH) immunoreactivity (IR) in the carotid body. A, Typical examples of the carotid body labeled by TH in a Ctrl and a prenatal nicotinic exposure (PNE) pup. Scale bars = 100 μM. In each inset (left), the area of the cross-section of the carotid body is surrounded by dashed white line, whereas the individuals of regions expressing TH are circled by solid white lines. B, TH-IR positive ratio = sum of regions expressing TH/the area of the cross-section of the carotid body × 100. C, Optical density of TH. n = 13 and 12 for Ctrl and PNE group. Mean ± SE; * and **P < .05 and P <.01, compared to Ctrl groups. PNE significantly reduces TH-IR positive ratio and TH expression in the carotid body.

Glomus cells’ population of the carotid body

The cross-section area and the sum areas were selected as shown in Figure 3A. TH-IR positive ratio was calculated from the sum of regions expressing TH/the area of the cross-section of the carotid body × 100.

Optical density of immunoreactivity

The optical density of TH-IR, NK1R-IR, and α7nAChR-IR in the carotid body were measured after transforming the images into the gray mode and quantified with the Adobe Photoshop 7.0 (Adobe, San Jose, CA). The quantification of these images was performed by technical staff blinded to the origin of the treatment. In assessing density, the mean optical density of the labeling was measured in a defined area (TH-IR positive area) with subtraction from the background in the same section (Hadjimarkou et al., 2009).

Data acquisition and statistical analysis

Raw data for tidal volume (VT), respiratory frequency (fR), and minute ventilation (VE) were all recorded by PowerLab/8sp (model ML 785; ADInstruments Inc., Colorado Springs, CO) and a computer with the LabChart Pro 7 software. The peak ventilatory response within 1–2 min of hypoxia and the decline phase of the response during the last minute of exposure were collected. The former was utilized to reflect the carotid body-mediated response and the latter to represent the sum of the excitatory (from the carotid body) and inhibitory (from the central nerve system) effects on HVR. HCVR data were collected during the last minute of exposure. Both responses were presented as a Δ% change from the baseline values. VE baseline values and optical densities of immunoreactivities were presented as absolute values. The relative changes in mRNA and TH-IR positive ratio were also expressed as % changes. Group data were reported as means ± SE. Student’s group t tests were used to analyze the significant differences between the 2 groups (Ctrl vs PNE). The Kolmogorov–Smirnov test was conducted to confirm the normality distribution of the data group followed by the Student’s t test. For the data with no negative values, the Student’s t test was repeated based on log transformed data which slightly improved the normality distribution and found no major difference in P values. For those data showing severe deviation from a normal distribution (Figure 1A ΔVT) or containing negative values (ΔfR in Figures 2 and 6B), we further applied non-parametric test (Rank-Sum test) and found no change in conclusion. Thus, results from Student’s t test based on untransformed data were presented in this study. Two-way ANOVA with repeated measures was used to analyze the significant differences among Ctrl and PNE before and after carotid body ablation or DOM treatment. When an overall test was significant, Tukey’s test was utilized for specific comparisons between individual groups. P values < .05 were considered significant.

FIG. 1.

FIG. 1.

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Peak (hypoxia for 1–2 minutes, A) and decline (the fifth min, B) phase of hypoxic ventilatory responses in Ctrl and PNE pups before and after carotid body ablation (CBA). Prenatal nicotinic exposure (PNE) significantly lowers both phases of minute ventilation (VE) and respiratory frequency (fR) without effect on tidal volume (VT) in the intact pups. The PNE-induced depression of VE and fR responses to hypoxia disappears after CBA. Mean ± SE; n = 10 for each group before CBA (the intact) and n = 5 and 8 for Ctrl and PNE pups after CBA. *P < .05.

FIG. 2.

FIG. 2.

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Ventilatory responses to hypercapnia (7% CO2) in Ctrl and prenatal nicotinic exposure (PNE) pups before and after carotid body ablation (CBA). A–C, PNE significantly lowers minute ventilation (VE) and respiratory frequency (fR) without effect on tidal volume (VT) in the intact pups. After CBA, PNE-induced decrease in VE and fR during hypercapnia disappears. Mean ± SE; n = 10 for each group before CBA (the intact), and n = 5 and 8 for Ctrl and PNE pups after CBA. *P < .05. After CBA, VE and fR were significantly reduced in Ctrl pups (P < .01).

FIG. 6.

FIG. 6.

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Prenatal nicotinic exposure (PNE) effects on the ventilator responses to intracarotid injection of SP (A, n = 7/group) and AR-R17779 (B, n = 5/group). The VE and fR responses to intracarotid injection of SP and AR-R17779 are significantly lower in the PNE than Ctrl pups. Mean ± SE; *P < .05, compared to Ctrl group.

RESULTS

Carotid Body Ablation Eliminated the PNE-Induced HVR/HCVR Depression

PNE significantly reduced HVR and HCVR in the intact pups, primarily due to the blunted fR response without a change in VT response. Neither PNE nor carotid body ablation (2 days after surgery) significantly changed baseline values of VE, fR, and VT (Table 1). Compared to the intact results, the peak HVR was reduced by 70% (149.5 ± 9.2% vs 36.4 ± 7.5%; P < .01) and the decline phase of HVR disappeared at the fifth min hypoxia (66.5 ± 6.4% vs −11.1 ± 6.7%; P < .01) after carotid body ablation in Ctrl pups. HCVR was diminished by 33% (123.5 ± 13.4% vs 82.2 ± 16.1%, P < .05) after carotid body ablation. The greatly reduced HVR and moderately diminished HCVR by carotid body ablation in Ctrl pups of this study are similar to previous reports (Gautier et al., 1993; Mouradian et al., 2012; Serra et al., 2001). Surprisingly, PNE had no effects on both HVR and HCVR after carotid body ablation (as illustrated in Figures 1 and 2), demonstrating an adverse impact of PNE on the carotid body.

TABLE 1.

The Baseline of Respiratory Variables in Conscious Animals

Substance Before CBA
 After CBA
Ctrl PNE Ctrl CB PNE CB
VE (ml/min.g) 1.38 ± 0.11 1.39 ± 0.06 1.56 ± 0.43 1.20 ± 0.13
fR (breaths/min) 122.8 ± 8.5 134.4 ± 4.6 131.2 ± 23.7 115.0 ± 10.1
VT (ml/kg) 10.8 ± 0.5 10.5 ± 0.6 11.5 ± 0.7 12.8 ± 1.3
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PNE and carotid body ablation do not alter the baseline of VE and fR, and VT. Mean ± SE, n = 10 per group before CBA and 5 and 8 for Ctrl and PNE pups after CBA.

PNE Decreased Carotid Body Glomus Cells/TH-IR Density

Previous studies have shown that the ratio of the sum areas with TH expression over the cross-section area of the carotid body reflects the relative population of glomus cells (Yamaguchi et al., 2003), whereas the optical density of TH-IR in the carotid body indicates the TH biosynthetic level in glomus cells. To study PNE adverse impacts on the carotid body, we compared the ratio and the optical density between the Ctrl and PNE pups. As exhibited in Figures 3A and B, the ratio was significantly reduced by PNE, pointing to a decrease in the numbers of glomus cells. Furthermore, the optical density of TH-IR in the carotid body was also diminished by PNE, suggesting a weakened ability of the carotid body to biosynthesize TH after PNE.

PNE Downregulated Carotid Body NK1R and α7nAChR Expression

We also detected mRNA levels of NK1R, α7nAChR, and DA2R in the carotid body and found that PNE significantly downregulated mRNA levels of NK1R and α7nAChR without a change of DA2R (Figure 4). To confirm PNE effect on NK1R and α7nAChR expression, the density of NK1R-IR and α7nAChR-IR in the carotid body was compared between Ctrl and PNE pups. The optical density of NK1R-IR, but not α7nAChR-IR, was significantly weakened by PNE (Figure 5).

FIG. 4.

FIG. 4.

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Prenatal nicotinic exposure (PNE) impacts on mRNA levels of neurokinin-1 receptor (NK1R) (A), α7 nicotinic acetylcholine receptor (α7nAChR) (B), and dopamine 2 receptor (DA2R) (C) in the carotid body. PNE significantly downregulates mRNA NK1R and α7nAChR but not DA2R. Mean ± SE; n = 7 in each group. *P < .05, compared to Ctrl group.

FIG. 5.

FIG. 5.

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Effects of prenatal nicotinic exposure (PNE) on neurokinin-1 receptor (NK1R)-IR and α7 nicotinic acetylcholine receptor (α7nAChR-IR) in the carotid body. A, Typical examples of NK1R-IR (top) and α7nAChR-IR (bottom) expression in the carotid body of Ctrl and PNE pups. Scale bars = 100 μM. Group data show that PNE significantly downregulates the optical density of NK1R-IR (B) but not α7nAChR-IR (C) in the carotid body. n = 10 in Ctrl and PNE group for NK1R and 7 and 6 in Ctrl and PNE group for α7nAChR. *P < .05, compared to Ctrl groups.

PNE Attenuated NK1R and α7nAChR activation-Induced Respiratory Responses

To confirm the functional significance of the change of NK1R and α7nAChR, we tested the ventilatory responses to intracarotid injection of substance P (a selective agonist for NK1R) or AR-R17779 (a selective agonist for α7nAChR) in anesthetized and spontaneously breathing Ctrl and PNE pups. As shown in Figure 6, intracarotid injection of substance P and AR-R17779 evoked remarkable VE augmentation via increasing fR. However, the evoked ventilatory response (particularly fR response) was significantly attenuated by PNE. The baseline values of VE, fR, and VT in the anesthetized preparation were not different between Ctrl and PNE pups (Table 2). In both Ctrl and PNE pups, intracarotid injection of the same volume of saline did not significantly alter baseline values (P > .05 compared to baseline “0” or between Ctrl and PNE), ie., VE (7.6 ± 5.7% vs 5.9 ± 3.8%), fR (3.1 ± 1.6% vs 4.5 ± 2.8%), and VT (4.2 ± 1.8% vs 4.7 ± 2.1%).

TABLE 2.

The Baseline of Respiratory Variables in Anesthetized Animals (Before Administration of SP and AR-R17779)

VE (ml/min.g)
 fR (breaths/min)
 VT (ml/kg)
Groups Ctrl PNE Ctrl PNE Ctrl PNE
SP 0.94 ± 0.11 0.98 ± 0.07 84.5 ± 7.3 88.1 ± 4.6 10.9 ± 0.7 10.8 ± 0.4
AR-R17779 1.17 ± 0.06 0.73 ± 0.13 81.8 ± 5.2 82.8 ± 8.3 10.6 ± 0.8 9.4 ± 2.0
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PNE does not alter the baseline of respiratory variables in anesthetized animals. n = 7/group for substance P and 5/group for AR-R17779.

Blockade of Peripheral DA2R Did Not Affect the PNE-Induced Depressed HVR

As shown in Figure 7, PNE decreased the VE, fR, and VT responses to hypoxia and this depression was not affected by intracarotid injection of DOM (a DA2R antagonist). In addition, DOM failed to strikingly alter baseline values of VE, VT, and fR in both Ctrl and PNE pups (Table 3).

FIG. 7.

FIG. 7.

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Hypoxic ventilatory responses (10% O2) to intracarotid injection of domperidone (DOM) in Ctrl and prenatal nicotinic exposure (PNE) pups (n = 6/group). DOM administration did not significantly change the VE (A), fR (B), and VT (C) responses to hypoxia in both Ctrl and PNE pup. PNE significantly decreased the hypoxic responses of VE, fR, and VT. Mean ± SE; *P < .05, compared between corresponding Ctrl and PNE.

TABLE 3.

The Baseline of Respiratory Variables in Anesthetized Animals for DOM Test

VE (ml/min.g)
 fR (breaths/min)
 VT (ml/kg)
Groups Ctrl PNE Ctrl PNE Ctrl PNE
Before DOM 0.73 ± 0.08 0.78 ± 0.04 78.7 ± 5.1 87.8 ± 8.7 9.4 ± 0.8 9.1 ± 0.5
After DOM 0.86 ± 0.07 0.88 ± 0.06 92.0 ± 5.1 113.7 ± 12.5 9.3 ± 0.6 8.0 ± 0.4
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DOM does not alter the baseline values of VE, fR, and VT in anesthetized pups before DOM treatment. Mean ± SE, n = 6 per group.

DISCUSSION

HVR and HCVR are most important in maintaining a mammal’s life by properly supplying enough oxygen to vital organs and regulate blood pH (Huang et al., 2010). The traditional PNE model showed the controversial impact on HVR and HCVR [ie., either mildly (Eugenin et al., 2008) or unchanged HVR and HCVR (Bamford and Carroll, 1999; Robinson et al., 2002) in neonates]. In this study, the “full term” PNE model covering the full gestation and postnatal period up to P12–14 (Zhuang et al., 2014) attenuated not only HVR by ∼35% but also HCVR by 33%. The lack of a constant appearance of HVR and HCVR depression in previous studies may be related to a significantly higher level of accumulated nicotinic exposure observed in our “full term” PNE than that in the traditional PNE (Zhuang et al., 2014).

HVR (2 min after the initial hypoxia) is known to be the sum of peripheral carotid body stimulation (Ang et al., 1992; Prabhakar, 2000; Vovk et al., 2004) and central nerve system depression (van Beek et al., 1984; Yelmen, 2004). The carotid body is also involved in generating HCVR (Carroll, 2003; Gauda and Lawson, 2000). The ability of nicotine to cross the blood-brain barrier to suppress respiration (Mitchell et al., 1963) raises a fundamental question as to what extent HVR and HCVR depression by PNE relies on the integrity of the carotid body. If the ventilatory depression is fully mediated by the central nerve system, a lower HVR /HCVR in PNE than Ctrl pups would be persistent after carotid body ablation. However, we found that carotid ablation lowered the peak HVR by 70% and HCVR by 35% 2 days after surgery with no longer difference between Ctrl and PNE pups. Our finding provides the first evidence indicating a key role of the dysfunction of the carotid body in depressing HVR and HCVR, although we cannot rule out possible impact of PNE on the central pathways of the carotid body. Ventilation and the activity of the carotid sinus nerve in response to hypoxia and hypercapnia were not significantly changed by traditional PNE in P3, 8, and 18 rat pups (Bamford and Carroll, 1999). This discrepancy may result from the insufficiency of PNE (see above) and the failure to target the postnatal period (P12–14), a developmental time-window vulnerable to induce cardiorespiratory disorders or failure (Gao et al., 2011). In fact, the nerve development at this time-window is equivalent to newborn infants at 2–4 month when SIDS often occurs (Ballanyi, 2004). Several factors are known to alter ventilation, including blood gases and metabolism (Vco2). However, both the HVR and HCVR depression induced by PNE in the present study are independent of these factors because they were not changed by PNE (Zhuang et al., 2014).

There are several lines of evidence in this study showing an adverse impact of PNE on the carotid body. First, PNE significantly shrank the TH-positive area, a semiquantitative analysis to estimate PNE impact on the relative population of glomus cells, consistent with a decreased number of glomus cells in the carotid body of some SIDS victims (Cole et al., 1979). A decrease in carotid body volume was also observed in SIDS victims (Naeye et al., 1976). However, the difficulty in isolating the intact carotid body from the surrounding tissues in rat pups limited our ability to perform such an experiment in this study. Second, the density of TH-IR was significantly lower in PNE than Ctrl pups. This points to a reduction of carotid body sensitivity by PNE because a lower TH expression has been reported to correlate with a poorer hypoxic response (Chai et al., 2011; Yamaguchi et al., 2003). Third, NK1R and α7nAChR mRNA levels and NK1R-IR expression in the carotid body were significantly decreased by PNE, but α7nAChR-IR was not affected by PNE. These 2 receptors are the major excitatory signal transducers in the carotid body (Nurse, 2010; Stephan-Blanchard et al., 2013) in control of the activity of carotid sinus nerve and respiratory output (Cragg et al., 1994; Nurse, 2010; Shirahata et al., 1998). Similar to a previous study with traditional PNE (Gauda et al., 2001), we found no significant change in mRNA DA2R after PNE in this study, suggesting an inability of PNE (no matter traditional or “full term”) to alter DA2R in the carotid body. Fourth, the respiratory responses to intracarotid injection of substance P (an agonist for NK1R) or AR-R17779 (an agonist for α7nAChR) were significantly higher in Ctrl than those in PNE pups, mainly via the depressing fR response. Interestingly, PNE significantly diminished the optical density of NK1R-IR, but not α7nAChR-IR. One may ask why there was a markedly depressed ventilatory response to intracarotid injection of AR-R17779 in PNE pups that failed to exhibit a significant change in carotid body α7nAChR-IR. Previous studies have demonstrated desensitization of α7nAChR after chronic nicotinic exposure (Clark et al., 2013; Sokolova et al., 2005). Thus, the weakened respiratory response to AR-R17779 in this study may be accountable for the possible desensitization of α7nAChR by PNE. In fact, lack of DOM effect on HVR (12% O2) has been previously observed (Julien et al., 2011). Collectively, our above findings support that PNE is able to cause an abnormality of NK1R and α7nAChR in the carotid body responsible for HVR and HCVR depression with limited effect on DA2R and endogenous dopamine. Our above findings support that PNE is able to cause an abnormality of NK1R and α7nAChR in the carotid body responsible for HVR and HCVR depression. The mechanism by which PNE downregulates NK1R in the carotid body is unclear. So far as we know, the direct effect of nicotine on NK1R has not been investigated. However, cigarette smoke reportedly promotes substance P release from pulmonary C-fibers (Xu and Xu, 2010). Therefore, it seems possible that PNE may elevate substance P level in the carotid body via stimulating C-fibers within the carotid sinus nerve to downregulate NK1R expression in the carotid body as a compensatory response.

There are several limitations in the present study. First, cigarette smoke contains numerous compounds emitted as gases and condensed tar particles, although nicotine is the major neurotoxic chemical (Kalra et al., 2002). We cannot rule out the possibility that compared to PNE, prenatal smoking exposure may have more potent and complicated impacts on HVR and HCVR. Second, the carotid body contains, in addition to NK1R, α7nAChR, and DA2R, other excitatory or inhibitory receptors responsible for HVR and HCVR, such as ATP/purinergic P2X receptors, 5-HT/5-HT2A and 5-HT3, GABA/GABAAR, and adenosine (Nurse, 2010). Their involvement in the PNE-induced HVR and HCVR depression is undetermined until further study. Third, we are not able to determine if the endogenous substance P, nicotine, and dopamine (the ligands of NK1R, α7nAChR, and DA2R) were affected by PNE because of the small size of the carotid body in pups. Fourth, owing to the 5 min hypoxia used in this study, we cannot deny a central involvement in depressed ventilatory response by PNE to the prolonged hypoxia. Acutely, PNE inhibitory effect on medullary respiratory-related nuclei has also been reported (Stephan-Blanchard et al., 2013).

In summary, our data show that “full term” PNE-induced significant depression of HVR and HCVR is dependent on the carotid body associated with the remarkable decrease in a number of TH-labeled glomus cells, TH-IR, and NK1R-IR expression and mRNA NK1R and α7nAChR in the carotid body. Moreover, PNE blunts ventilatory responses to intracarotid injection of substance P and AR-R17779. Establishment of PNE adverse impacts on the carotid body by downregulating TH and NK1R expression and possibly desensitizing α7nAChR advances our recognition of the nicotinic toxic impact on the carotid body. Given that the severity of HVR and HCVR depression by PNE is correlated to the subsequent death (Zhuang et al., 2014), the present results broaden our understanding of the pathogenesis of the respiratory failure in SIDS victims and benefit potential therapeutic treatments to reduce or prevent the respiratory failure. To delineate the direct effect of PNE on the carotid body, further studies are required to determine the reduced sensitivity of the carotid sinus nerve in response to hypoxia and hypercapnia after PNE and the roles of abnormal carotid body NK1R and α7nAChR after PNE in mediating these responses.

ACKNOWLEDGMENTS

The authors are grateful to Ellen Blake for editing and Dr Shuguang Leng for his statistical assistant.

FUNDING

This study was supported in part by the National Institutes of Health grants (HL-107462 and HL-119683 to F.X. and HL-96914 to L.Y.L.).

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