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Background Continuous positive airway pressure (CPAP) is a primary mode of respiratory support for preterm infants. Animal studies have shown long-term detrimental effects on lung/airway development, particularly airway (AW) hyper-reactivity, as an unfortunate consequence of neonatal CPAP. Since the hyaluronan (HA) synthesizing enzyme hyaluronan synthase-3 (HAS3) is involved in various adult pulmonary disorders, the present study used a neonatal mouse model to investigate the role of HAS3 in CPAP-induced AW hyper-reactivity. Methods Male and female neonatal mice were fitted with a custom-made mask for delivery of daily CPAP 3 h/day for 7 days. At postnatal day 21 (2 weeks after CPAP ended), airway (AW) hyper-reactivity and HAS3 expression were assessed with and without in vitro HAS3 siRNA treatment. Results MRIs of 3-day-old mice confirmed that CPAP increased lung volume with incrementing inflation pressures. CPAP increased AW reactivity in both male and female mice, which was associated with increased airway smooth muscle and epithelial HAS3 immunoreactivity. CPAP did not affect HA accumulation, but HAS3 siRNA reversed CPAP-induced AW hyper-reactivity and reduced HAS3 expression. Conclusions These data in mice implicate a role for HAS3 in long-term effects of CPAP in the developing airway in the context of preterm birth and CPAP therapy. Impact Neonatal CPAP increases airway smooth muscle and epithelial HAS3 expression in mice. CPAP-induced airway hyper-reactivity is modulated by HAS3. These data enhance our understanding of the role mechanical forces play on lung development. These data are a significance step toward understanding CPAP effects on developing airway. These data may impact clinical recognition of the ways that CPAP may contribute to wheezing disorders of former preterm infants.

Background Continuous positive airway pressure (CPAP) in preterm infants is initially beneficial, but animal models suggest longer term detrimental airway effects towards asthma. We used a neonatal CPAP mouse model and human fetal airway smooth muscle (ASM) to investigate the role of extracellular calcium-sensing receptor (CaSR) in these effects. Methods Newborn wild type and smooth muscle-specific CaSR-/- mice were given CPAP for 7 days via a custom device (mimicking CPAP in premature infants), and recovered in normoxia for another 14 days (representing infants at 3-4 years). Airway reactivity was tested using lung slices, and airway CaSR quantified. Role of CaSR was tested using NPS2143 (inhibitor) or siRNA in WT mice. Fetal ASM cells stretched cyclically with/without static stretch mimicking breathing and CPAP were analyzed for intracellular Ca2+ ([Ca2+]i) responses, role of CaSR, and signaling cascades. Results CPAP increased airway reactivity in WT but not CaSR-/- mice, increasing ASM CaSR. NPS2143 or CaSR siRNA reversed CPAP effects in WT mice. CPAP increased fetal ASM [Ca2+]I, blocked by NPS2143, and increased ERK1/2 and RhoA suggesting two mechanisms by which stretch increases CaSR. Conclusions These data implicate CaSR in CPAP effects on airway function with implications for wheezing in former preterm infants. Impact Neonatal CPAP increases airway reactivity to bronchoconstrictor agonist. CPAP increases smooth muscle expression of the extracellular calcium-sensing receptor (CaSR). Inhibition or absence of CaSR blunts CPAP effects on contractility. These data suggest a causal/contributory role for CaSR in stretch effects on the developing airway. These data may impact clinical recognition of the ways that CPAP may contribute to wheezing disorders of former preterm infants.

Continuous infusion of prostaglandin E1 (PGE1) is used to maintain ductus arteriosus patency in infants with critical congenital heart disease, but it can also cause central apnea suggesting an effect on respiratory neural control. In this study, we investigated whether 1) PGE1inhibits the various phases of the acute hypoxic ventilatory response (HVR; an index of respiratory control dysfunction) and increases apnea incidence in neonatal rats; and 2) whether these changes would be reversible with caffeine pretreatment. Whole body plethysmography was used to assess the HVR and apnea incidence in neonatal rats 2 h following a single bolus intraperitoneal injection of PGE1with and without prior caffeine treatment. Untreated rats exhibited a biphasic HVR characterized by an initial increase in minute ventilation followed by a ventilatory decline of the late phase (~5th minute) of the HVR. PGE1had a dose-dependent effect on the HVR. Contrary to our hypothesis, the lowest dose (1 µg/kg) of PGE1prevented the ventilatory decline of the late phase of the HVR. However, PGE1tended to increase postsigh apnea incidence and the coefficient of variability (CV) of breathing frequency, suggesting increased respiratory instability. PGE1also decreased brainstem microglia mRNA and increased neuronal nitric oxide synthase (nNOS) and platelet-derived growth factor-β (PDGF-β) gene expression. Caffeine pretreatment prevented these effects of PGE1, and the adenosine A2Areceptor inhibitor MSX-3 had similar preventative effects. Prostaglandin appears to have deleterious effects on brainstem respiratory control regions, possibly involving a microglial-dependent mechanism. The compensatory effects of caffeine or MSX-3 treatment raises the question of whether prostaglandin may also operate on an adenosine-dependent pathway.

Infants born with neonatal opioid withdrawal syndrome (NOWS) can display abnormal cardiorespiratory patterns including tachypnea, tachycardia, and impaired ventilatory responses to hypoxia (HVR) and hypercapnia (HCVR). Chronic morphine exposure is associated with increased midbrain microglial expression. Using a rat model of pre- and post-natal morphine exposure, we assessed cardiorespiratory features of NOWS (resting tachycardia and tachypnea) including the attenuated HVR and HCVR and whether they are associated with increased brainstem microglia expression. Pregnant rats (dams) received twice-daily subcutaneous injections of morphine (5 mg/kg) during the third (last) week of pregnancy to simulate 3rd trimester in utero opioid exposure. Offspring then received once-daily subcutaneous injections of morphine (0.5 mg/kg) until postnatal (P) day P10 days of age to simulate postnatal morphine therapy. Cardiorespiratory responses were assessed 24 h later (P11 days) following spontaneous withdrawal. Compared to saline-treated pups, morphine-exposed offspring exhibited tachycardia and tachypnea as well as an attenuated HVR and HCVR. Microglial cell counts were increased in the nucleus tractus solitarius (nTS), dorsal motor nucleus of the vagus (DMNV) and nucleus ambiguous (NAamb), but not the retrapezoid nucleus (RTN) or the non-cardiorespriatory region, the cuneate nucleus (CN). These data suggest that the cardiorespiratory features and autonomic dysregulation in NOWS infants may be associated with altered microglial function in specific brainstem cardiorespiratory control regions.

Airway smooth muscle (ASM) regulation of airway structure and contractility is critical in fetal/neonatal physiology in health and disease. Fetal lungs experience higher Ca(2+) environment which may impact on extracellular Ca(2+) ([Ca(2+)](o)) sensing receptor (CaSR). Well-known in parathyroid gland, CaSR is also expressed in late embryonic lung mesenchyme. Using cells from 18–22 week human fetal lungs, we tested the hypothesis that CaSR regulates intracellular Ca(2+) ([Ca(2+)](i)) in fetal ASM (fASM). Compared to adult ASM, CaSR expression was higher in fASM, while fluorescence Ca(2+) imaging showed that [Ca(2+)](i) was more sensitive to altered [Ca(2+)](o). fASM [Ca(2+)](i) responses to histamine were also more sensitive to [Ca(2+)](o) (0–2 mM) compared to adult, enhanced by calcimimetic R568 but blunted by calcilytic NPS2143. [Ca(2+)](i) was enhanced by endogenous CaSR agonist spermine (again higher sensitivity compared to adult). Inhibition of phospholipase C (U73122; siRNA) or IP(3) receptor (Xestospongin C) blunted [Ca(2+)](o) sensitivity and R568 effects. NPS2143 potentiated U73122 effects. Store-operated Ca(2+) entry was potentiated by R568. Traction force microscopy showed responsiveness of fASM cellular contractility to [Ca(2+)](o) and NPS2143. Separately, fASM proliferation showed sensitivity to [Ca(2+)](o) and NPS2143. These results demonstrate functional CaSR in developing ASM that modulates airway contractility and proliferation.

Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification, airway hyperreactivity, and pulmonary hypertension. In our BPD model, we have investigated the metabolism of the bronchodilator and pulmonary vasodilator GSNO (S-nitrosoglutathione). We have shown the GSNO catabolic enzyme encoded by adh5 (alcohol dehydrogenase-5), GSNO reductase, is epigenetically upregulated in hyperoxia. Here, we investigated the distribution of GSNO reductase expression in human BPD and created an animal model that recapitulates the human data. Blinded comparisons of GSNO reductase protein expression were performed in human lung tissues from infants and children with and without BPD. BPD phenotypes were evaluated in global (adh5(−/−)) and conditional smooth muscle (smooth muscle/adh5(−/−)) adh5 knockout mice. GSNO reductase was prominently expressed in the airways and vessels of human BPD subjects. Compared with controls, expression was greater in BPD smooth muscle, particularly in vascular smooth muscle (2.4-fold; P = 0.003). The BPD mouse model of neonatal hyperoxia caused significant alveolar simplification, airway hyperreactivity, and right ventricular and vessel hypertrophy. Global adh5(−/−) mice were protected from all three aspects of BPD, whereas smooth muscle/adh5(−/−) mice were only protected from pulmonary hypertensive changes. These data suggest adh5 is required for the development of BPD. Expression in the pulmonary vasculature is relevant to the pathophysiology of BPD-associated pulmonary hypertension. GSNO-mimetic agents or GSNO reductase inhibitors, both of which are currently in clinical trials for other conditions, could be considered for further study in BPD.

Supplemental O2 (hyperoxia), necessary for maintenance of oxygenation in premature infants, contributes to neonatal and pediatric airway diseases including asthma. Airway smooth muscle (ASM) is a key resident cell type, responding to hyperoxia with increased contractility and remodeling [proliferation, extracellular matrix (ECM) production], making the mechanisms underlying hyperoxia effects on ASM significant. Recognizing that fetal lungs experience a higher extracellular Ca2+ ([Ca2+]o) environment, we previously reported that the calcium sensing receptor (CaSR) is expressed and functional in human fetal ASM (fASM). In this study, using fASM cells from 18 to 22 week human fetal lungs, we tested the hypothesis that CaSR contributes to hyperoxia effects on developing ASM. Moderate hyperoxia (50% O2) increased fASM CaSR expression. Fluorescence [Ca2+]i imaging showed hyperoxia increased [Ca2+]i responses to histamine that was more sensitive to altered [Ca2+]o, and promoted IP3 induced intracellular Ca2+ release and store-operated Ca2+ entry: effects blunted by the calcilytic NPS2143. Hyperoxia did not significantly increase mitochondrial calcium which was regulated by CaSR irrespective of oxygen levels. Separately, fASM cell proliferation and ECM deposition (collagens but not fibronectin) showed sensitivity to [Ca2+]o that was enhanced by hyperoxia, but blunted by NPS2143. Effects of hyperoxia involved p42/44 ERK via CaSR and HIF1α. These results demonstrate functional CaSR in developing ASM that contributes to hyperoxia-induced contractility and remodeling that may be relevant to perinatal airway disease.

Perinatal inflammation and infection are commonly associated with various respiratory morbidities in preterm infants including apnea of prematurity. In this study, we investigated whether pulmonary inflammation via intra-tracheal micro-injection of lipopolysaccharide (LPS) into neonatal rats modifies respiratory neural control via an IL-1β receptor-dependent mechanism. Prior to an intra-tracheal micro-injection of LPS (1mg/kg), 10day old (Postnatal age, P10) rats received an intraperitoneal (i.p.) or intracisternal (i.c.) micro-injection of the IL-1β receptor antagonist AF12198. Whole-body plethysmography was performed two hours later to assess the magnitude of the acute hypoxic (HVR) and hypercapnic (HCVR) ventilatory responses. Intra-tracheal LPS dose-dependently attenuated the acute HVR compared to saline (control) treated rats, whereas the HCVR was not affected. Pre-treatment with an i.c. (but not i.p.) micro-injection of AF12198 15min prior to LPS prevented the attenuated HVR. These data indicate that intrapulmonary inflammation affects brainstem respiratory neural pathways mediating the ventilatory response to acute hypoxia via an IL-1β-dependent pathway. These findings are relevant to our understanding of the way that pulmonary inflammation may affect central neural mechanisms of respiratory insufficiency commonly seen in preterm infants.

Acute Lung Injury (ALI) alters pulmonary reflex responses, in part due to changes in modulation within the lung and airway neuronal control networks. We hypothesized that synaptic efficacy of nucleus tractus solitarii (nTS) neurons, receiving input from lung, airway, and other viscerosensory afferent fibers, would decrease following ALI. Sprague Dawley neonatal rats (postnatal days 9-11) were given intratracheal installations of saline or bleomycin (a well-characterized model that reproduces the pattern of ALI) and then, one week later, in vitro slices were prepared for whole-cell and perforated whole-cell patch-clamp experiments (postnatal days 16-21). In preparations from ALI rats, 2nd-order nTS neurons had significantly decreased amplitudes of both spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs), compared to saline controls. Rise and decay times of sEPSCs were slower in whole-cell recordings from ALI animals. Similarly, the amplitude of tractus solitarii evoked EPSCs (TS-eEPSCs) were significantly lower in 2nd-order nTS neurons from ALI rats. Overall these results suggest the presence of postsynaptic depression at TS-nTS synapses receiving lung, airway, and other viscerosensory afferent tractus solitarii input after bleomycin-induced ALI.

Key points Neonatal sustained hypoxia exposure modifies brainstem microglia and serotonin expression. The altered brainstem neurochemistry is associated with impaired ventilatory responses to acute hypoxia and mortality. The deleterious effects of sustained hypoxia exposure can be prevented by an inhibitor of activated microglia. These observations demonstrate a potential cause of the brainstem serotonin abnormalities thought to be involved in sudden infant death syndrome. Abstract We showed previously that the end of the second postnatal week (days P11–15) represents a period of development during which the respiratory neural control system exhibits a heightened vulnerability to sustained hypoxia (SH, 11% O2, 5 days) exposure. In the current study, we investigated whether the vulnerability to SH during the same developmental time period is associated with changes in brainstem serotonin (5-HT) expression and whether it can be prevented by the microglia inhibitor minocycline. Using whole-body plethysmography, SH attenuated the acute (5 min) hypoxic ventilatory response (HVR) and caused a high incidence of mortality compared to normoxia rats. SH also increased microglia cell numbers and decreased 5-HT immunoreactivity in the nucleus of the solitary tract (nTS) and dorsal motor nucleus of the vagus (DMNV). The attenuated HVR, mortality, and changes in nTS and DMNV immunoreactivity was prevented by minocycline (25 mg kg−1/2 days during SH). These data demonstrate that the 5-HT abnormalities in distinct respiratory neural control regions can be initiated by prolonged hypoxia exposure and may be modulated by microglia activity. These observations share several commonalities with the risk factors thought to underlie the aetiology of sudden infant death syndrome, including: (1) a vulnerable neonate; (2) a critical period of development; (3) evidence of hypoxia; (4) brainstem gliosis (particularly the nTS and DMNV); and (5) 5-HT abnormalities.

Moderate hyperoxic exposure in preterm infants contributes to subsequent airway dysfunction and to risk of developing recurrent wheeze and asthma. The regulatory mechanisms that can contribute to hyperoxia-induced airway dysfunction are still under investigation. Recent studies in mice show that hyperoxia increases brain-derived neurotrophic factor (BDNF), a growth factor that increases airway smooth muscle (ASM) proliferation and contractility. We assessed the mechanisms underlying effects of moderate hyperoxia (50% O2) on BDNF expression and secretion in developing human ASM. Hyperoxia increased BDNF secretion, but did not alter endogenous BDNF mRNA or intracellular protein levels. Exposure to hyperoxia significantly increased [Ca2+]i responses to histamine, an effect blunted by the BDNF chelator TrkB-Fc. Hyperoxia also increased ASM cAMP levels, associated with reduced PDE4 activity, but did not alter protein kinase A (PKA) activity or adenylyl cyclase mRNA levels. However, 50% O2 increased expression of Epac2, which is activated by cAMP and can regulate protein secretion. Silencing RNA studies indicated that Epac2, but not Epac1, is important for hyperoxia-induced BDNF secretion, while PKA inhibition did not influence BDNF secretion. In turn, BDNF had autocrine effects of enhancing ASM cAMP levels, an effect inhibited by TrkB and BDNF siRNAs. Together, these novel studies suggest that hyperoxia can modulate BDNF secretion, via cAMP-mediated Epac2 activation in ASM, resulting in a positive feedback effect of BDNF-mediated elevation in cAMP levels. The potential functional role of this pathway is to sustain BDNF secretion following hyperoxic stimulus, leading to enhanced ASM contractility and proliferation.

Background: Wheezing disorders are prominent in former preterm infants beyond the neonatal period. Objectives: We used a neonatal mouse model to investigate the time course of airway hyperreactivity in response to mild (40% oxygen) or severe (70% oxygen) neonatal hyperoxia. Methods: After hyperoxic exposure during the first week of postnatal life, we measured changes in airway reactivity using the in vitro living lung slice preparation at the end of exposure [postnatal day 8 (P8)] and 2 weeks later (P21). This was accompanied by measures of smooth muscle actin, myosin light chain (MLC) and alveolar morphology. Results: Neither mild nor severe hyperoxia exposure affected airway reactivity to methacholine at P8 compared to normoxic controls. In contrast, airway reactivity was enhanced at P21 in mice exposed to mild (but not severe) hyperoxia, 2 weeks after exposure ended. This was associated with increased airway α-smooth muscle actin expression at P21 after 40% oxygen exposure without a significant increase in MLC. Alveolar morphology via radial alveolar counts was comparably diminished by both 40 and 70% oxygen at both P8 and P21. Conclusions: These data demonstrate that early mild hyperoxia exposure causes a delayed augmentation of airway reactivity, suggesting a long-term alteration in the trajectory of airway smooth muscle development and consistent with resultant symptomatology.

Acute intermittent hypoxia (AIH; three 5-min hypoxic episodes) causes a form of phrenic motor facilitation (pMF) known as phrenic long-term facilitation (pLTF); pLTF is initiated by spinal activation of Gq protein-coupled 5-HT2 receptors. Because α1 adrenergic receptors are expressed in the phrenic motor nucleus and are also Gq protein-coupled, we hypothesized that α1 receptors are sufficient, but not necessary for AIH-induced pLTF. In anesthetized, paralyzed, and ventilated rats, episodic spinal application of the α1 receptor agonist phenylephrine (PE) elicited dose-dependent pMF (10 and 100 μM, P < 0.05; but not 1 μM). PE-induced pMF was blocked by the α1 receptor antagonist prazosin (1 mM; −20 ± 20% at 60 min, −5 ± 21% at 90 min; n = 6). Although α1 receptor activation is sufficient to induce pMF, it was not necessary for AIH-induced pLTF because intrathecal prazosin (1 mM) did not alter AIH-induced pLTF (56 ± 9% at 60 min, 78 ± 12% at 90 min; n = 9). Intravenous (iv) prazosin (150 μg/kg) appeared to reduce pLTF (21 ± 9% at 60 min, 26 ± 8% at 90 min), but this effect was not significant. Hypoglossal long-term facilitation was unaffected by intrathecal prazosin, but was blocked by iv prazosin (−4 ± 14% at 60 min, −13 ± 18% at 90 min), suggesting different LTF mechanisms in different motor neuron pools. In conclusion, Gq protein-coupled α1 adrenergic receptors evoke pMF, but they are not necessary for AIH-induced pLTF.

Neonatal chronic intermittent hypoxia (CIH) enhances the ventilatory sensitivity to acute hypoxia (acute hypoxic ventilatory response, HVR), whereas sustained hypoxia (SH) can have the opposite effect. Therefore, we investigated whether neonatal rats pre-treated with SH prior to CIH exhibit a modified HVR. Rat pups were exposed to CIH (5% O2/5 min, 8 h/day) between 6 and 15 days of postnatal age (P6-15) after pre-treatment with either normoxia or SH (11% O2; P1-5). Using whole-body plethysmography, the acute (5 min, 10% O2) HVR at P16 (1 day post-CIH) was unchanged following CIH (67.9 ± 6.7% above baseline) and also SH (58.8 ± 10.5%) compared to age-matched normoxic rats (54.7 ± 6.3%). In contrast, the HVR was attenuated (16.5 ± 6.0%) in CIH exposed rats pre-treated with SH. These data suggest that while neonatal SH and CIH alone have little effect on the magnitude of the acute HVR, their combined effects impose a synergistic disturbance to postnatal development of the HVR. These data could provide important insight into the consequences of not maintaining adequate levels of oxygen saturation during the early neonatal period, especially in vulnerable preterm infants susceptible to frequent bouts of hypoxemic events (CIH) that are commonly associated with apnea of prematurity.

Evidence of respiratory abnormalities and vulnerability to infection during a critical period of development have been implicated in Sudden Infant Death Syndrome (SIDS). Here we investigated whether the acute hypoxic ventilatory response (HVR) exhibits a heightened vulnerability to the endotoxin lipopolysaccharide (LPS) during a critical period of development. The acute HVR was measured 2h after an i.p. injection of saline or LPS (0.1mg/kg) at various postnatal (P) ages (P5, P10, or P20days). LPS attenuated the early (1-2min) and late (4-6min) phase of the acute HVR in P10 but not P5 or P20 rats. The P10 age group exhibited the largest increase in brainstem TNFα and iNOS mRNA expression following LPS. LPS also caused a higher mortality rate in P10 rats (48%) compared to P5 (12%) and P20 (0%) age groups. After stratifying LPS treated P10 rats into survivors vs non-survivors, only the latter exhibited an attenuated HVR (specifically the early phase). Thus, the heightened vulnerability to endotoxin exposure during this critical period of development is characterized by a depression of the ventilatory response to acute hypoxia in association with an increased incidence of mortality. These data share similarities with some of the circumstances surrounding a SIDS scenario, including evidence of infection, increased brainstem cytokine expression, a disturbance in respiratory control, and a peak incidence of mortality during a critical period of development.

Acute intermittent hypoxia (AIH) facilitates phrenic motor output by a mechanism that requires spinal serotonin (type 2) receptor activation, NADPH oxidase activity and formation of reactive oxygen species (ROS). Episodic spinal serotonin (5-HT) receptor activation alone, without changes in oxygenation, is sufficient to elicit NADPH oxidase-dependent phrenic motor facilitation (pMF). Here we investigated: (1) whether serotonin 2A and/or 2B (5-HT2A/B) receptors are expressed in identified phrenic motor neurons, and (2) which receptor subtype is capable of eliciting NADPH-oxidase-dependent pMF. In anesthetized, artificially ventilated adult rats, episodic C4 intrathecal injections (3×6 μl injections, 5 min intervals) of a 5-HT2A (DOI) or 5-HT2B (BW723C86) receptor agonist elicited progressive and sustained increases in integrated phrenic nerve burst amplitude (i.e. pMF), an effect lasting at least 90 min post-injection for both receptor subtypes. 5-HT2A and 5-HT2B receptor agonist-induced pMF were both blocked by selective antagonists (ketanserin and SB206553, respectively), but not by antagonists to the other receptor subtype. Single injections of either agonist failed to elicit pMF, demonstrating a need for episodic receptor activation. Phrenic motor neurons retrogradely labeled with cholera toxin B fragment expressed both 5-HT2A and 5-HT2B receptors. Pre-treatment with NADPH oxidase inhibitors (apocynin and diphenylenodium (DPI)) blocked 5-HT2B, but not 5-HT2A-induced pMF. Thus, multiple spinal type 2 serotonin receptors elicit pMF, but they act via distinct mechanisms that differ in their requirement for NADPH oxidase activity.

The effects of lowering body temperature (Tb) on metabolic rate, ventilation, and the strength of the Hering-Breüer expiratory promoting reflex (HB reflex; determined from an inhibitory ratio calculated from volumetric measurements of the respiratory rhythm) were examined in 18-day-old ectothermic pouch young of the tammar wallaby during normoxia or hypoxia (10% O2). Hypoxia and hypothermia, either singularly or combined, depressed metabolic rate. At all Tb, the hypoxic hyperventilation was associated with a significant hyperpnea. At pouch Tb (36.5°C) during normoxia, inflation of the lungs with -5 or -10 cmH2O extrathoracic pressure induced a significant HB reflex. Exposure to cold reduced the strength of the reflex, almost abolishing it at 28°C. For Tb above 28°C, the reflex in hypoxia was always less than the corresponding normoxic value. Taken in context with the changes in metabolic state that occurred, these data in the ectothermic marsupial newborn suggest that the decline in the HB reflex during moderate hypothermia is the result of a direct effect of Tb on vagal mechanisms rather than a temperature-driven decline in metabolic rate that should have acted to strengthen the HB reflex. Therefore, it seems that inputs inhibitory to breathing are more negatively affected during cold than those inputs that are excitatory.

We investigated whether pre-treatment with neonatal sustained hypoxia (SH) prior to chronic intermittent hypoxia (SH+CIH) would modify in vitro carotid body (CB) chemoreceptor activity and the excitability of neurons in the caudal nucleus of the solitary tract (nTS). Sustained hypoxia followed by CIH exposure simulates an oxygen paradigm experienced by extremely premature infants who developed persistent apnea. Rat pups were treated with 5 days of SH (11% O2) from postnatal age 1 (P1) followed by 10 days of subsequent chronic intermittent hypoxia (CIH, 5% O2/5 min, 8 h/day, between P6 and P15) as described previously (Mayer et al., Respir. Physiol. Neurobiol. 187(2): 167-75, 2013). At the end of SH+CIH exposure (P16), basal firing frequency was enhanced, and the hypoxic sensory response of single unit CB chemoafferents was attenuated. Further, basal firing frequency and the amplitude of evoked excitatory post-synaptic currents (ESPC's) of nTS neurons was augmented compared to age-matched rats raised in normoxia. These effects were unique to SH+CIH exposure as neither SH or CIH alone elicited any comparable effect on chemoafferent activity or nTS function. These data indicated that pre-treatment with neonatal SH prior to CIH exposure uniquely modified mechanisms of peripheral (CB) and central (nTS) neural function in a way that would be expected to disturb the ventilatory response to acute hypoxia.

Wheezing is a major long-term respiratory morbidity in preterm infants with and without bronchopulmonary dysplasia. We hypothesized that mild vs. severe hyperoxic exposure in neonatal mice differentially affects airway smooth muscle hypertrophy and resultant airway reactivity. Newborn mice were exposed to 7 days of mild (40% oxygen) or severe (70% oxygen) hyperoxia vs. room air controls. Respiratory system resistance (Rrs), compliance (Crs), and airway reactivity were measured 14 days after oxygen exposure ended under ketamine/xylazine anesthesia. Baseline Rrs increased and Crs decreased in both treatment groups. Methacholine challenge dose dependently increased Rrs and decreased Crs in 40% oxygen-exposed mice, whereas Rrs and Crs responses were similar between 70% oxygen-exposed and normoxic controls. Airway smooth muscle thickness was increased in 40%- but not 70%-exposed mice, whereas collagen increased and both alveolar number and radial alveolar counts decreased after 40% and 70% oxygen. These data indicate that severity of hyperoxia may differentially affect structural and functional changes in the developing mouse airway that contribute to longer-term hyperreactivity. These findings may be important to our understanding of the complex role of neonatal supplemental oxygen therapy in postnatal development of airway responsiveness.

Sustained hypoxia (SH) exposure during a vulnerable period of neonatal development (~11-15 days of postnatal (P) age) attenuates the acute hypoxic (HVR) ventilatory response in rats. In the current study, we investigated whether the effects of SH exposure on the acute HVR varies among different rat strains. Sprague-dawley (SD), Fisher (F), and Spontaneously Hypertensive (SHR) rats were exposed to SH (11% O2, 5 days) starting at P11. The acute HVR and Dejours test were assessed using whole-body plethysmography the day after (P16) SH exposure ended. SH abolished the HVR in SHR, but not SD or F rats compared to normoxia raised animals. The transient ventilatory depression following brief (5 minutes) 100% O2 exposure (Dejours test) was also absent in SHR rats following SH treatment. These data indicate a strain difference in the vulnerability of the respiratory system to neonatal SH exposure; possible genetic determinants for this phenomenon requires further study.

Sustained hypoxia exposure during a finite window of neonatal development (~10-15 days of postnatal (P) age) attenuates the acute hypoxic (HVR) and hypercapnic (HCVR) ventilatory response in rats. In the current study, we investigated whether the HVR and HCVR are also modified by LPS exposure during a similar period of development. Rat pups received an intraperitoneal injection of either saline or 0.1mg/kg of LPS at either P5, P10, or P20; the acute HVR and HCVR were measured using whole-body plethysmography ~2.5hrs later. LPS attenuated the HVR (but not the HCVR) in P10 rats (1.8 ml/g/min) compared to saline (2.4 ml/g/min) treated rats, whereas younger (P5) or older (P10) rats were unaffected. LPS was lethal in some rats at some time within 24 hours after LPS; P10 pups appeared particularly vulnerable with a higher rate of mortality (27% of pups) than P5 (9% of pups) or P20 (0% of pups) treated rats. These data reveal the presence of a developmental window (~P10) that may be at a heightened level of vuln...

The premature transition from fetal to neonatal life is accompanied by an immature respiratory neural control system. Most preterm infants exhibit recurrent apnea, resulting in repetitive oscillations in O(2) saturation (intermittent hypoxia, IH). Numerous factors are likely to play a role in the etiology of apnea including inputs from the carotid chemoreceptors. Despite major advances in our understanding of carotid chemoreceptor function in the early neonatal period, however, their contribution to the initiation of an apneic event and its eventual termination are still largely speculative. Recent findings have provided a detailed account of the postnatal changes in the incidence of hypoxemic events associated with apnea, and there is anecdotal evidence for a positive correlation with carotid chemoreceptor maturation. Furthermore, studies on non-human animal models have shown that chronic IH sensitizes the carotid chemoreceptors, which has been proposed to perpetuate the occurrence of apnea. An alternative hypothesis is that sensitization of the carotid chemoreceptors could represent an important protective mechanism to defend against severe hypoxemia. The purpose of this review, therefore, is to discuss how the carotid chemoreceptors may contribute to the initiation and termination of an apneic event in the neonate and the use of xanthine therapy in the prevention of apnea.

We tested the hypotheses that: (1) long-term facilitation (LTF) following acute intermittent hypoxia (AIH) varies among three inbred rat strains: Fischer 344 (F344), Brown Norway (BN) and Lewis rats and (2) ventral cervical spinal levels of genes important for phrenic LTF (pLTF) vary in association with pLTF magnitude. Lewis and F344, but not BN rats exhibited significant increases in phrenic and hypoglossal burst amplitude 60min post-AIH that were significantly greater than control experiments without AIH, indicating strain differences in phrenic (98%, 56% and 20%, respectively) and hypoglossal LTF (66%, 77% and 5%, respectively). Ventral spinal 5-HT(2A) receptor mRNA and protein levels were higher in F344 and Lewis versus BN, suggesting that higher 5-HT(2A) receptor levels are associated with greater pLTF. More complex relationships were found for 5-HT(7), BDNF and TrkB mRNA. BN had higher 5-HT(7) and TrkB mRNA versus F344; BN and Lewis had higher BDNF mRNA levels versus F344. Genetic variations in serotonergic function may underlie strain differences in AIH-induced pLTF.

Acute intermittent hypoxia (AIH) elicits a form of respiratory plasticity known as long-term facilitation (LTF). LTF is a progressive and sustained increase in respiratory motor output as expressed in phrenic and hypoglossal (XII) nerve activity. Since reactive oxygen species (ROS) play important roles in several forms of neuroplasticity, and ROS production is increased by intermittent hypoxia, we tested the hypothesis that ROS are necessary for phrenic and hypoglossal LTF following AIH. Urethane anesthetized, paralyzed, vagotomized and pump ventilated Sprague Dawley rats were exposed to AIH (11% O2, 3, 5 min episodes, 5 min intervals), and both phrenic and XII nerve activity were monitored for 60 min post-AIH. Although phrenic and XII LTF were observed in control rats, intravenous Manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), a superoxide anion scavenger, attenuated both phrenic and XII LTF in a dose dependent manner. Localized application of MnTMPyP (5.5mM; 10µl) to the intrathecal space of the cervical spinal cord (C4) abolished phrenic, but not XII LTF. Thus, ROS are necessary for AIH-induced respiratory LTF, and the relevant ROS appear to be localized near respiratory motor nuclei since cervical MnTMPyP injections impaired phrenic (and not XII) LTF. Phrenic LTF is a novel form of ROS-dependent neuroplasticity since its ROS-dependence resides in the spinal cord.