Introduction: Hyaluronan (HA) is a major component of the extracellular matrix. Increased pulmonary HA concentrations are associated with several respiratory disorders and is a pathophysiological feature of lung disease. We investigated whether elevated urine HA is a biomarker of an unfavorable 40-week respiratory outcome in preterm infants. Methods: Infants comprised a cohort of preterm neonates <31 weeks gestational age (GA) from the Prematurity-Related Ventilatory Control (Pre-Vent) multicenter study. HA was quantified in urine obtained at 1 week and 1 month of age. Respiratory status at 40 weeks post-menstrual age (PMA) was classified as unfavorable [either (1) deceased at or before 40 weeks PMA, (2) an inpatient on respiratory medication, O-2 or other respiratory support at 40 weeks, or (3) discharged prior to 40 weeks on medications/O-2/other respiratory support], or favorable (alive and previously discharged, or inpatient and off respiratory medications, off O-2, and off other respiratory support at 40 weeks PMA). The association between urine HA and the unfavorable 40 week PMA outcome was examined using a multivariate logistic generalized estimation equation model. Results: Infants with higher HA at 1 week (but not 1 month) showed increased odds of unfavorable respiratory outcome at 40 weeks PMA (OR [95% CI] = 1.87 per 0.01 mg [1.27, 2.73]). Discussion and Conclusion: Neonatal urine screening for HA could identify infants at risk for death or need for respiratory support at term-corrected age (40 weeks PMA). The relationship between elevated HA at 1 week and an unfavorable 40 week outcome was stronger in infants with lower GA.

BACKGROUND:Hypoxemia is a physiological manifestation of immature respiratory control in preterm neonates, which is likely impacted by neurotransmitter imbalances. We investigated relationships between plasma levels of the neurotransmitter serotonin (5-HT), metabolites of tryptophan (TRP), and parameters of hypoxemia in preterm neonates. METHODS:TRP, 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and kynurenic acid (KA) were analyzed in platelet-poor plasma at ~1 week and ~1 month of life from a prospective cohort of 168 preterm neonates <31 weeks gestational age (GA). Frequency of intermittent hypoxemia (IH) events and percent time hypoxemic (<80%) were analyzed in a 6 h window after the blood draw. RESULTS:At 1 week, infants with detectable plasma 5-HT had fewer IH events (OR (95% CI) = 0.52 (0.29, 0.31)) and less percent time <80% (OR (95% CI) = 0.54 (0.31, 0.95)) compared to infants with undetectable 5-HT. A similar relationship occurred at 1 month. At 1 week, infants with higher KA showed greater percent time <80% (OR (95% CI) = 1.90 (1.03, 3.50)). TRP, 5-HIAA or KA were not associated with IH frequency at either postnatal age. IH frequency and percent time <80% were positively associated with GA < 29 weeks. CONCLUSIONS:Circulating neuromodulators 5-HT and KA might represent biomarkers of immature respiratory control contributing to hypoxemia in preterm neonates. IMPACT:Hypoxemia events are frequent in preterm infants and are associated with poor outcomes. Mechanisms driving hypoxemia such as immature respiratory control may include central and peripheral imbalances in modulatory neurotransmitters. This study found associations between the plasma neuromodulators serotonin and kynurenic acid and parameters of hypoxemia in preterm neonates. Imbalances in plasma biomarkers affecting respiratory control may help identify neonates at risk of short- and long-term adverse outcomes.

Continuous positive airway pressure (CPAP) is a primary non-invasive mode of respiratory support for preterm infants. However, emerging evidence suggests CPAP could be an underlying contributor to the unintended pathophysiology of wheezing and associated airway hyperreactivity (AHR) in former preterm infants. The therapeutic benefits of mesenchymal stem cells (MSCs) have been demonstrated in a variety of animal models and several clinical trials are currently underway to assess their safety profiles in the setting of prematurity and bronchopulmonary dysplasia (BPD). In the present study, using a mouse model of neonatal CPAP, we investigated whether conditioned medium harvested from cultures of human bone-marrow derived mesenchymal stem cells (hMSC) could rescue the CPAP-induced AHR, based upon previous observations of their anti-AHR properties. Newborn mice (male and female) were fitted with a custom-made mask for delivery of daily CPAP 3 h/day for the first 7 postnatal days. At postnatal day 21 (two weeks after CPAP ended), lungs were removed, precisioncut lung slices were sectioned and incubated for 48 h in vitro in conditioned medium collected from cultures of three different hMSC donors. As expected, CPAP resulted in AHR to methacholine compared to untreated control mice. hMSC conditioned medium from the cultures of all three donors completely reversed AHR. These data reveal potential therapeutic benefits of hMSC therapy, which may be capable of rescuing the long-term adverse effects of neonatal CPAP on human airway function.

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.

Former preterm infants, many of whom required supplemental O-2 support, exhibit sleep disordered breathing and attenuated ventilatory responses to acute hypoxia (HVR) beyond their NICU stay. There is an increasing awareness that early detection of biomarkers in biological fluids may be useful predictors/identifiers of short-and long-term morbidities. In the present study, we identified serotonin (5-HT), dopamine (DA) and hyaluronan (HA) as three potential biomarkers that may be increased by neonatal hyperoxia and tested whether they would be associated with an impaired HVR in a rat model of supplemental O-2 exposure. Neonatal rats (postnatal age (P) 6 days, P6) exposed to hyperoxia (40% FIO2, 24 h/day between P1-P5 days of age) exhibited an attenuated early (1 min), but not the late (4 -5 min) phase of the HVR compared to normoxia control rats; the attenuated early phase HVR was associated with increased levels of DA (urine and serum), 5-HT (platelet poor plasma only, PPP), and HA (serum only). At P21, both the early and late phases of the HVR were attenuated, but serum and urine levels of all 3 biomarkers were similar to age-matched control rats. These data indicate that changes in several serum and/or urine biomarkers (5-HT, DA, and HA) following short-term (days) neonatal hyperoxia can signify long-term (weeks) respiratory control dysfunction. Further studies are needed to determine whether early detection of similar biomarkers could be convenient predictors of increased risk of abnormalities in respiratory control including sleep disordered breathing in former preterm infants who had received prior supplemental O-2 and who might also be at increased risk of SIDS.

Moderate acute intermittent hypoxia (mAIH) elicits a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF). Preconditioning with modest protocols of chronic intermittent hypoxia enhances pLTF, demonstrating pLTF metaplasticity. Since "low-dose" protocols of repetitive acute intermittent hypoxia (rAIH) show promise as a therapeutic modality to restore respiratory (and nonrespiratory) motor function in clinical disorders with compromised breathing, we tested 1) whether preconditioning with a mild rAIH protocol enhances pLTF and hypoglossal (XII) LTF and 2) whether the enhancement is regulated by glycolytic flux. In anesthetized, paralyzed, and ventilated adult male Lewis rats, mAIH (three 5-min episodes of 10% O2) elicited pLTF (pLTF at 60 min post-mAIH: 49 ± 5% baseline). rAIH preconditioning (ten 5-min episodes of 11% O2/day with 5-min normoxic intervals, 3 times per week, for 4 wk) significantly enhanced pLTF (100 ± 16% baseline). XII LTF was unaffected by rAIH. When glycolytic flux was inhibited by 2-deoxy-d-glucose (2-DG) administered via drinking water (~80 mg·kg-1·day-1), pLTF returned to normal levels (58 ± 8% baseline); 2-DG had no effect on pLTF in normoxia-pretreated rats (59 ± 7% baseline). In ventral cervical (C4/5) spinal homogenates, rAIH increased inducible nitric oxide synthase mRNA vs. normoxic controls, an effect blocked by 2-DG. However, there were no detectable effects of rAIH or 2-DG on several molecules associated with phrenic motor plasticity, including serotonin 2A, serotonin 7, brain-derived neurotrophic factor, tropomyosin receptor kinase B, or VEGF mRNA. We conclude that modest, but prolonged, rAIH elicits pLTF metaplasticity and that a drug known to inhibit glycolytic flux (2-DG) blocks pLTF enhancement.

The extracellular matrix (ECM) modulates brain maturation and plays a major role in regulating neuronal plasticity during critical periods of development. We examined 1) whether there is a critical postnatal period of ECM expression in brain stem cardiorespiratory control regions and 2) whether the attenuated hypoxic ventilatory response (HVR) following neonatal sustained (5 days) hypoxia [SH (11% O 2 , 24 h/day)] exposure is associated with altered ECM formation. The nucleus tractus solitarius (nTS), dorsal motor nucleus of the vagus, hypoglossal motor nucleus, cuneate nucleus, and area postrema were immunofluorescently processed for aggrecan and Wisteria floribunda agglutinin (WFA), a key proteoglycan of the ECM and the perineuronal net. From postnatal day ( P) 5 ( P5), aggrecan and WFA expression increased postnatally in all regions. We observed an abrupt increase in aggrecan expression in the nTS, a region that integrates and receives afferent inputs from the carotid body, between P10 and P15 followed by a distinct and transient plateau between P15 and P20. WFA expression in the nTS exhibited an analogous transient plateau, but it occurred earlier (between P10 and P15). SH between P11 and P15 attenuated the HVR (assessed at P16) and increased aggrecan (but not WFA) expression in the nTS, dorsal motor nucleus of the vagus, and area postrema. An intracisternal microinjection of chondroitinase ABC, an enzyme that digests chondroitin sulfate proteoglycans, rescued the HVR and the increased aggrecan expression. These data indicate that important stages of ECM formation take place in key brain stem respiratory neural control regions and appear to be associated with a heightened vulnerability to hypoxia.

Daily acute intermittent hypoxia (dAIH) elicits respiratory plasticity, enhancing respiratory motor output and restoring breathing capacity after incomplete cervical spinal injuries (cSCI). We hypothesized that dAIH-induced functional recovery of breathing capacity would occur after both acute (2 weeks) and chronic (8 weeks) cSCI, but through distinct cellular mechanisms. Specifically, we hypothesized that dAIH-induced breathing recovery would occur through serotonin-independent mechanisms 2wks post C2 cervical hemisection (C2Hs), versus serotonin-dependent mechanisms 8wks post C2Hs. In two independent studies, dAIH or sham (normoxia) was initiated 1 week (Study 1) or 7 weeks (Study 2) post-C2Hs to test our hypothesis. Rats were pre-treated with intra-peritoneal vehicle or methysergide, a broad-spectrum serotonin receptor antagonist, to determine the role of serotonin signaling in dAIH-induced functional recovery. Our data support the hypothesis that dAIH-induced recovery of breathing capacity transitions from a serotonin-independent mechanism with acute C2Hs to a serotonin-dependent mechanism with chronic C2Hs. An understanding of shifting mechanisms giving rise to dAIH-induced respiratory motor plasticity is vital for clinical translation of dAIH as a therapeutic modality.

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.

Prior studies have demonstrated the existence of a critical window of neurochemical expression that occurs in the brainstem at approximately 12 days of postnatal (P12) age in the rat. The critical window is characterized by changes in the expression of various neurotransmitters, receptors, trophic factors, and metabolic enzymes in various brainstem respiratory control regions. Presumably these changes represent important neurodevelopmental events necessary for appropriate maturation of the cardio‐respiratory control system. However, studies from our lab have begun to demonstrate the critical window is a period of development during which the respiratory control system exhibits a heightened vulnerability to environmental challenges. For example, five days of sustained hypoxia exposure (SH: 11% O2) between P11–15 days, which encompasses the P12 critical window, abolished the ventilatory response to acute hypoxia (HVR (delta baseline): 0.63 ± 0.31 ml/g/min) compared to age‐matched rats raised in normoxia (0.12 ± 0.45 ml/g/min). The absence of a HVR in these rats was associated with an unexpected and substantial degree of mortality that occurred ~3 days later. These effects were unique to the critical window as neither younger (P1–5) or older (P21–25) rats were affected by the same exposure to SH. We have also shown the critical window appears to be an important stage of brainstem neurodevelopment because it is represented by a transient surge in constitutive expression of brainstem mRNA for NeuroD1 and CyclinB2. However, SH between P11–15 reduced their expression (NeuroD1: 0.66 ± 0.11; CyclinB2: 0.74 ± 0.14 fold‐change from normoxia control rats), indicating a major and potentially fatal disturbance in brainstem mechanisms of neurodevelopment. The critical window of development was also characterized by a transient increase in the constitutive expression of brainstem TNFα mRNA, which was further increased by SH exposure (1.47 ± 0.09 fold‐change from control). Further, SH also decreased serotonin (5‐HT) levels specifically within the dorsal motor nucleus of the vagus (DMNV) and nucleus of the solitary tract (nTS). We speculate that SH exposure interferes with key neurodevelopmental events, ultimately leading to a lethal disturbance in brainstem neurochemistry within distinct (cardio)–respiratory control regions and may involve a mechanism associated with an aberrant cytokine response. These data could contribute to our understanding of the pathways involved in the brainstem neurochemical abnormalities observed in Sudden Infant Death Syndrome (SIDS). Support or Funding Information Department of Pediatrics, Rainbow Babies & Children's Hospital, Case Western Reserve University

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% O-2, 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.

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% O-2) from postnatal age 1 (P1) followed by 10 days of subsequent chronic intermittent hypoxia (CIH, 5% O-2/5 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. Published by Elsevier B.V.

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.

Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF) by a mechanism that requires spinal serotonin (5-HT) receptor activation and NADPH oxidase (NOX) activity. Here, we investigated whether: (1) spinal nitric oxide synthase (NOS) activity is necessary for AIH-induced pLTF; (2) episodic exogenous nitric oxide (NO) is sufficient to elicit phrenic motor facilitation (pMF) without AIH (i.e. pharmacologically); and (3) NO-induced pMF requires spinal 5-HT2B receptor and NOX activation. In anesthetized, mechanically ventilated adult male rats, AIH (3 × 5-min episodes; 10% O2; 5 min) elicited a progressive increase in the amplitude of integrated phrenic nerve bursts (i.e. pLTF), which lasted 60 min post-AIH (45.1 ± 8.6% baseline). Pre-treatment with intrathecal (i.t.) injections of a neuronal NOS inhibitor (nNOS-inhibitor-1) near the phrenic motor nucleus attenuated pLTF (14.7 ± 2.5%), whereas an inducible NOS (iNOS) inhibitor (1400 W) had no effect (56.3 ± 8.0%). Episodic i.t. injections (3 × 5 μl volume; 5 min) of a NO donor (sodium nitroprusside; SNP) elicited pMF similar in time-course and magnitude (40.4 ± 6.0%, 60 min post-injection) to AIH-induced pLTF. SNP-induced pMF was blocked by a 5-HT2B receptor antagonist (SB206553), a superoxide dismutase mimetic (MnTMPyP), and two NOX inhibitors (apocynin and DPI). Neither pLTF nor pMF was affected by pre-treatment with a protein kinase G (PKG) inhibitor (KT-5823). Thus, spinal nNOS activity is necessary for AIH-induced pLTF, and episodic spinal NO is sufficient to elicit pMF by a mechanism that requires 5-HT2B receptor activation and NOX-derived ROS formation, which indicates AIH (and NO) elicits spinal respiratory plasticity by a nitrergic-serotonergic mechanism.

The first postnatal weeks represent a period of development in the rat during which the respiratory neural control system may be vulnerable to aberrant environmental stressors. In the present study, we investigated whether sustained hypoxia (SH; 11% O2) exposure starting at different postnatal ages differentially modifies the acute hypoxic (HVR) and hypercapnic ventilatory response (HCVR). Three different groups of rat pups were exposed to 5 days of SH, starting at either postnatal age 1 (SH1-5), 11 (SH11-15), or 21 (SH21-25) days. Whole body plethysmography was used to assess the HVR and HCVR the day after SH exposure ended. The primary results indicated that 1) the HVR and HCVR of SH11-15 rats were absent or attenuated (respectively) compared with age-matched rats raised in normoxia; 2) there was a profoundly high (∼84% of pups) incidence of unexplained mortality in the SH11-15 rats; and 3) these phenomena were unique to the SH11-15 group with no comparable effect of the SH exposure on the HVR, HCVR, or mortality in the younger (SH1-5) or older (SH21-25) rats. These results share several commonalities with the risk factors thought to underlie the etiology of sudden infant death syndrome, including 1) a vulnerable neonate; 2) a critical period of development; and 3) an environmental stressor.