Neonatal Intestinal Segmental Volvulus: What Are the Differences with Midgut Volvulus?

OBJECTIVE: Intestinal volvulus in the neonate is a surgical emergency caused by either midgut volvulus (MV) with intestinal malrotation or less commonly, by segmental volvulus (SV) without intestinal malrotation. The aim of our study was to investigate if MV and SV can be differentiated by clinical course, intraoperative findings, and postoperative outcomes. METHODS: Using a defined search strategy, two investigators independently identified all studies comparing MV and SV in neonates. PRISMA guidelines were followed, and a meta-analysis was performed using RevMan 5.3. RESULTS: Of 1,026 abstracts screened, 104 full-text articles were analyzed, and 3 comparative studies were selected (112 patients). There were no differences in gestational age (37 vs. 36 weeks), birth weight (2,989 vs. 2,712 g), and age at presentation (6.9 vs. 3.8 days). SV was more commonly associated with abnormal findings on fetal ultrasound (US; 65 vs. 11.6%; p < 0.00001). Preoperatively, SV was more commonly associated with abdominal distension (32 vs. 77%; p < 0.05), whereas MV with a whirlpool sign on ultrasound (57 vs. 3%; p < 0.01). Bilious vomiting had similar incidence in both (88 ± 4% vs. 50 ± 5%). Intraoperatively, SV had a higher incidence of intestinal atresia (2 vs. 19%; p < 0.05) and need for bowel resection (13 vs. 91%; p < 0.00001). There were no differences in postoperative complications (13% MV vs. 14% SV), short bowel syndrome (15% MV vs. 0% SV; data available only from one study), and mortality (12% MV vs. 2% SV). CONCLUSION: Our study highlights the paucity of studies on SV in neonates. Nonetheless, our meta-analysis clearly indicates that SV is an entity on its own with distinct clinical features and intraoperative findings that are different from MV. SV should be considered as one of the differential diagnoses in all term and preterm babies with bilious vomiting after MV was ruled out-especially if abnormal fetal US and abdominal distension is present.

L-citrulline attenuates lipopolysaccharide-induced inflammatory lung injury in neonatal rats.

BACKGROUND: Prenatal or postnatal lung inflammation and oxidative stress disrupt alveolo-vascular development leading to bronchopulmonary dysplasia (BPD) with and without pulmonary hypertension. L-citrulline (L-CIT), a nonessential amino acid, alleviates inflammatory and hyperoxic lung injury in preclinical models of BPD. L-CIT modulates signaling pathways mediating inflammation, oxidative stress, and mitochondrial biogenesis-processes operative in the development of BPD. We hypothesize that L-CIT will attenuate lipopolysaccharide (LPS)-induced inflammation and oxidative stress in our rat model of neonatal lung injury. METHODS: Newborn rats during the saccular stage of lung development were used to investigate the effect of L-CIT on LPS-induced lung histopathology and pathways involved in inflammatory, antioxidative processes, and mitochondrial biogenesis in lungs in vivo, and in primary culture of pulmonary artery smooth muscle cells, in vitro. RESULTS: L-CIT protected the newborn rat lung from LPS-induced: lung histopathology, ROS production, NFκB nuclear translocation, and upregulation of gene and protein expression of inflammatory cytokines (IL-1ß, IL-8, MCP-1α, and TNF-α). L-CIT maintained mitochondrial morphology, increased protein levels of PGC-1α, NRF1, and TFAM (transcription factors involved in mitochondrial biogenesis), and induced SIRT1, SIRT3, and superoxide dismutases protein expression. CONCLUSION: L-CIT may be efficacious in decreasing early lung inflammation and oxidative stress mitigating progression to BPD. IMPACT: The nonessential amino acid L-citrulline (L-CIT) mitigated lipopolysaccharide (LPS)-induced lung injury in the early stage of lung development in the newborn rat. This is the first study describing the effect of L-CIT on the signaling pathways operative in bronchopulmonary dysplasia (BPD) in a preclinical inflammatory model of newborn lung injury. If our findings translate to premature infants, L-CIT could decrease inflammation, oxidative stress and preserve mitochondrial health in the lung of premature infants at risk for BPD.

PGC-1α activity and mitochondrial dysfunction in preterm infants.

Extremely low gestational age neonates (ELGANs) are born in a relatively hyperoxic environment with weak antioxidant defenses, placing them at high risk for mitochondrial dysfunction affecting multiple organ systems including the nervous, respiratory, ocular, and gastrointestinal systems. The brain and lungs are highly affected by mitochondrial dysfunction and dysregulation in the neonate, causing white matter injury (WMI) and bronchopulmonary dysplasia (BPD), respectively. Adequate mitochondrial function is important in providing sufficient energy for organ development as it relates to alveolarization and axonal myelination and decreasing oxidative stress via reactive oxygen species (ROS) and reactive nitrogen species (RNS) detoxification. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is a master regulator of mitochondrial biogenesis and function. Since mitochondrial dysfunction is at the root of WMI and BPD pathobiology, exploring therapies that can regulate PGC-1α activity may be beneficial. This review article describes several promising therapeutic agents that can mitigate mitochondrial dysfunction through direct and indirect activation and upregulation of the PGC-1α pathway. Metformin, resveratrol, omega 3 fatty acids, montelukast, L-citrulline, and adiponectin are promising candidates that require further pre-clinical and clinical studies to understand their efficacy in decreasing the burden of disease from WMI and BPD in preterm infants.

Risk factors for prolonged mechanical ventilation in neonates following gastrointestinal surgery.

Background: Prolonged mechanical ventilation (MV) should be avoided in neonates. Noninvasive ventilation (NIV) can facilitate weaning from MV but has risks for patients immediately following foregut surgery due to the potential risk of anastomotic leak. We evaluated the risk factors for prolonged MV following intestinal surgery in neonates. Methods: We retrospectively reviewed 253 neonates undergoing intestinal surgery in 2017-2018 to identify risk factors for prolonged MV, and determine the correlation between NIV and anastomotic leak in a tertiary neonatal intensive care unit that performs the greatest number of neonatal surgeries in Ontario. Results: The most common diagnoses were necrotizing enterocolitis/spontaneous intestinal perforation (NEC/SIP) 21%, intestinal atresia 16%, esophageal atresia/tracheoesophageal fistula 14%, ano-rectal malformation 13%, malrotation/volvulus 11%, gastroschisis 9% and omphalocele 4%. The median (IQR) duration of MV post-surgery was 3 (1-8) days with 25.7 % (n=65) of neonates on MV for >7 days. Compared to infants on MV post-surgery for ≤7 days, those with MV>7 days were of lower gestational age, birth weight and weight at surgery, but a higher proportion underwent stoma creation, had a longer duration of opioid administration and higher rates of moderate to severe bronchopulmonary dysplasia (BPD) and mortality (P<0.05). Generalized linear regression analysis showed lower gestational age (GA) and longer opioid administration were associated with longer duration of MV (P<0.001), but indication for surgery, weight at surgery and stoma creation didn't correlate with longer duration of MV (P>0.05). Of the 122 patients handled by one-stage resection with primary anastomosis, 22.1% (n=27) received NIV with 74.1% (n=20) commenced on NIV after 7 days post-surgery, anastomotic leak was detected in 2.5 % (3/122) patients and didn't correlate with NIV. Conclusions: Lower GA and longer opioid administration were risk factors for prolonged MV in neonates following intestinal surgery. Further research is needed to investigate modifiable practices around pain assessment/ventilation in these patients, and the correlation between NIV and anastomotic leak.

Ileal lactobezoar in extreme premature infant complicated by intestinal perforation: A case report.

INTRODUCTION AND IMPORTANCE: Lactobezor is a rare complication that has been reported more in the stomach, however it may be located anywhere in the intestine. CASE REPORT: Reported here, is a case of ileal lactobezoar which was complicated by perforation and was mimicking necrotizing enterocolitis in presentation, ex preterm (26 weeks) male infant who presented at day of life 18th (2 days after BM fortification) with hemodynamic instability and intestinal perforation, which was diagnosed by Abdominal X-ray and Ultrasound necessitating urgent laparotomy. CLINICAL DISCUSSION: Laparotomy revealed an area of ileal perforation and an inspissated mass which was confirmed to be lactobezoar by pathology, ileostomy was performed. The baby had an acute postoperative status of hypovolemic shock which was managed clinically, then was restarted on feeds, and the stoma was reversed 9 weeks later. CONCLUSION: Lactobezoar, although rare, but numbers increased especially with the rise in numbers of extremely preterm infants worldwide, it most commonly presents later in life but in some cases, such as our case it may happen in 1st 2-3 weeks after birth and may cause significant complications as perforation making its differentiation from common GI problems in neonates as NEC more challenging.

Remote ischemic conditioning in necrotizing enterocolitis: study protocol of a multi-center phase II feasibility randomized controlled trial.

PURPOSE: Remote ischemic conditioning (RIC) is a maneuver involving brief cycles of ischemia reperfusion in an individual's limb. In the early stage of experimental NEC, RIC decreased intestinal injury and prolonged survival by counteracting the derangements in intestinal microcirculation. A single-center phase I study demonstrated that the performance of RIC was safe in neonates with NEC. The aim of this phase II RCT was to evaluate the safety and feasibility of RIC, to identify challenges in recruitment, retainment, and to inform a phase III RCT to evaluate efficacy. METHODS: RIC will be performed by trained research personnel and will consist of four cycles of limb ischemia (4-min via cuff inflation) followed by reperfusion (4-min via cuff deflation), repeated on two consecutive days post randomization. The primary endpoint of this RCT is feasibility and acceptability of recruiting and randomizing neonates within 24 h from NEC diagnosis as well as masking and completing the RIC intervention. RESULTS: We created a novel international consortium for this trial and created a consensus on the diagnostic criteria for NEC and protocol for the trial. The phase II multicenter-masked feasibility RCT will be conducted at 12 centers in Canada, USA, Sweden, The Netherlands, UK, and Spain. The inclusion criteria are: gestational age < 33 weeks, weight ≥ 750 g, NEC receiving medical treatment, and diagnosis established within previous 24 h. Neonates will be randomized to RIC (intervention) or no-RIC (control) and will continue to receive standard management of NEC. We expect to recruit and randomize 40% of eligible patients in the collaborating centers (78 patients; 39/arm) in 30 months. Bayesian methods will be used to combine uninformative prior distributions with the corresponding observed proportions from this trial to determine posterior distributions for parameters of feasibility. CONCLUSIONS: The newly established NEC consortium has generated novel data on NEC diagnosis and defined the feasibility parameters for the introduction of a novel treatment in NEC. This phase II RCT will inform a future phase III RCT to evaluate the efficacy and safety of RIC in early-stage NEC.

SARS-CoV-2 Associated Respiratory Failure in a Preterm Infant and the Outcome after Remdesivir Treatment.

Severe coronavirus disease 2019 (COVID-19) occurs in approximately 10% of neonates infected with severe acute respiratory syndrome coronavirus 2. Guidelines for optimal management of severe COVID-19 in neonates do not exist. In this report, we describe a late-preterm neonate with severe COVID-19, requiring invasive mechanical ventilation who recovered following treatment with remdesivir and high dose dexamethasone.

Recombinant adiponectin protects the newborn rat lung from lipopolysaccharide-induced inflammatory injury.

Preterm infants are at high risk for developing bronchopulmonary dysplasia and pulmonary hypertension from inflammatory lung injury. In adult models, adiponectin (APN)-an adipocyte-derived hormone-protects the lung from inflammatory injury and pulmonary vascular remodeling. Cord blood APN levels in premature infants born < 26 weeks gestation are 5% of the level in infants born at term. We previously reported the expression profile of APN and its receptors in neonatal rat lung homogenates during the first 3 weeks of postnatal development. Here, we characterize the expression profile of APN and its receptors in specific lung cells and the effects of exogenous recombinant APN (rAPN) on lipopolysaccharide-(LPS)-induced cytokine and chemokine production in total lung homogenates and specific lung cells. In vitro, rAPN added to primary cultures of pulmonary artery smooth muscle cells attenuated the expression of LPS-induced pro-inflammatory cytokines while increasing the expression of anti-inflammatory cytokines. In vivo, intraperitoneal rAPN (2 mg/kg), given 4 hr prior to intrapharyngeal administration of LPS (5 mg/kg) to newborn rats at postnatal day 4, significantly reduced gene and protein expression of the pro-inflammatory cytokine IL-1ß and reduced protein expression of the chemokines monocyte chemoattractant protein (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1α) in the lung. LPS-induced histopathological changes in the lung were also decreased. Moreover, rAPN given 20 hr after intrapharyngeal LPS had a similar effect on lung inflammation. These findings suggest a role for APN in protecting the lung from inflammation during early stages of lung development.

Could Postnatal Age-Related Uridine Diphosphate Glucuronic Acid Be a Rate-Limiting Factor in the Metabolism of Morphine During the First Week of Life?

Neonates experience dramatic changes in the disposition of drugs after birth as a result of enzyme maturation and environmental adjustment, challenging therapeutic decision making. In this research, we establish postnatal age, postmenstrual age, and body weight as physiologically reasonable predictors of morphine's clearance in neonates. By integrating knowledge of bilirubin, morphine, and other drugs metabolized by glucuronidation pathways from previously published studies, we hypothesize that uridine diphosphate glucuronic acid, a postnatal age-dependent sugar, plays an important role in the metabolism of morphine during the first week of life. This finding can be extended to other drugs metabolized by uridine diphosphate glucuronosyltransferase pathways in neonates and thus has important clinical implications for the use of drugs in this population.

Chronic Intermittent Hypoxia in Premature Infants: The Link Between Low Fat Stores, Adiponectin Receptor Signaling and Lung Injury.

Premature infants have chronic intermittent hypoxia (CIH) that increases morbidity, and the youngest and the smallest premature infants are at the greatest risk. The combination of lung injury from inflammation/oxidative stress causing low functional residual capacity combined with frequent short apneas leads to CIH. Adiponectin (APN) is an adipose-derived adipokine that protects the lung from inflammation and oxidative stress. Premature and small for gestational age (SGA) infants have minimal body fat and low levels of circulating APN. To begin to understand the potential role of APN in lung protection during lung development, we characterized the developmental profile of APN and APN receptors (AdipoR1 and AdipoR2) protein and mRNA expression in the newborn rat lung at fetal day (FD) 19, and postnatal days (PD) 1, 4, 7, 10, 14, 21, and 28. Protein levels in lung homogenates were measured by western blot analyses; relative mRNA expression was detected by quantitative PCR (qPCR); and serum high molecular weight (HMW) APN was measured using enzyme-linked immunosorbent assay (ELISA). Results: APN protein and mRNA levels were lowest at FD19 and PD1, increased 2.2-fold at PD4, decreased at PD10, and then increased again at PD21. AdipoR1 protein and mRNA levels peaked at PD1, followed by a threefold drop by PD4, and remained low until PD21. AdipoR2 protein and mRNA levels also peaked at PD1, but remained high at PD4, followed by a 1.7-fold drop by PD10 that remained low by PD21. Serum APN levels detected by ELISA did not differ from PD4 to PD28. To date, this is the first report characterizing APN and APN receptor protein and mRNA expression in the rat lung during development. The developmental stage of the newborn rat lung models that of the premature human infant; both are in the saccular stage of lung development. In the newborn rat lung, alveolarization begins at PD4, peaks at PD10, and ends at PD21. Importantly, we found that AdipoR1 receptor protein and mRNA expression is lowest during lung alveolarization (PD4 to PD21). Thus, we speculate that low levels of AdipoR1 during lung alveolarization contributes to the increased susceptibility to developing acute lung edema and chronic lung injury such as bronchopulmonary dysplasia (BPD) in premature human infants.

Contribution of relative leptin and adiponectin deficiencies in premature infants to chronic intermittent hypoxia: Exploring a new hypothesis.

Chronic intermittent hypoxia (CIH) occurs frequently in premature infants who have apnea of prematurity. Immaturity of the respiratory network from low central respiratory drive and the greater contribution of the carotid body on baseline breathing leads to respiratory instability in premature infants presenting as apnea and periodic breathing. During the 2nd week after birth, the smallest and the youngest premature infants have increased frequency of apnea and periodic breathing and associated oxygen desaturations that can persist for weeks after birth. CIH increases the production of reactive oxygen species that causes tissue damage. Premature infants have decreased capacity to scavenge reactive oxygen species. Oxidative injury is the cause of many of the co-morbidities that are seen in premature infants. In this review we discuss who low fat mass and the resulting relative deficiencies in leptin and adiponectin could contribute to the increase frequency of oxygen desaturations that occurs days after birth in the smallest and youngest premature infants. Leptin is a central respiratory stimulant and adiponectin protects the lung from vascular leak, oxidative injury and vascular remodeling.

Mucous fistula refeeding decreases parenteral nutrition exposure in postsurgical premature neonates.

BACKGROUND/PURPOSE: Premature neonates can develop intraabdominal conditions requiring emergent bowel resection and enterostomy. Parenteral nutrition (PN) is often required, but results in cholestasis. Mucous fistula refeeding allows for functional restoration of continuity. We sought to determine the effect of refeeding on nutrition intake, PN dependence, and PN associated hepatotoxicity while evaluating the safety of this practice. METHODS: A retrospective review of neonates who underwent bowel resection and small bowel enterostomy with or without mucous fistula over 2years was undertaken. Patients who underwent mucous fistula refeeding (RF) were compared to those who did not (OST). Primary outcomes included days from surgery to discontinuation of PN and goal enteral feeds, and total days on PN. Secondary outcomes were related to PN hepatotoxicity. RESULTS: Thirteen RF and eleven OST were identified. There were no significant differences among markers of critical illness (p>0.20). In the interoperative period, RF patients reached goal enteral feeds earlier than OST patients (median 28 versus 43days; p=0.03) and were able to have PN discontinued earlier (median 25 versus 41days; p=0.04). Following anastomosis, the magnitude of effect was more pronounced, with RF patients reaching goal enteral feeds earlier than OST patients (median 7.5 versus 20days; p≤0.001) and having PN discontinued sooner (30.5 versus 48days; p=0.001). CONCLUSIONS: RF neonates reached goal feeds and were able to be weaned from PN sooner than OST patients. A prospective multicenter trial of refeeding is needed to define the benefits and potential side effects of refeeding in a larger patient population in varied care environments.

Mechanisms of modulation of cytokine release by human cord blood monocytes exposed to high concentrations of caffeine.

BACKGROUND: Serum caffeine concentrations >20 µg/ml (100 µmol/l) in infants treated for apnea of prematurity increases TNF-α and decreases IL-10, changes that perhaps are linked to comorbidities. We hypothesize that this proinflammatory cytokine profile may be linked to differential binding of caffeine to adenosine receptor subtypes (AR), inhibition of phosphodiesterases (PDEs), and modulation of toll-like receptors (TLR). METHODS: Lipopolysaccharide-activated cord blood monocytes (CBM) from 19 infants were exposed to caffeine (0-200 µmol/l) with or without previous exposure to A1R, A3R, or PDE IV antagonists to determine changes in dose-response curves. Cytokines levels (enzyme-linked immunosorbent assay (ELISA)), intracellular cyclic adenosine monophosphate (cAMP) accumulation (enzyme immunoassay (EIA)), and TLR gene expression (real time qRT PCR) were measured. RESULTS: Caffeine at ≤100 µmol/l decreased TNF-α levels (~25%, P = 0.01) and cAMP. All caffeine concentrations decreased IL-10 levels (17-35%, P < 0.01). A1R, A3R, and PDE blockades decreased TNF-α (31, 21, and 88%, P ≤ 0.01), but not IL-10. Caffeine further decreased TNF-α following A3R and PDE blockades. Caffeine concentrations directly correlated to TLR4 gene expression (r = 0.84; P < 0.001). CONCLUSION: Neither A3R, nor PDE blockades are involved in caffeine's modulation of cytokine release by CBM at any concentration. Besides A1R blockade, caffeine's upregulation of TLR4 may promote inflammation at high concentrations.

Revisiting cAMP signaling in the carotid body.

Chronic carotid body (CB) activation is now recognized as being essential in the development of hypertension and promoting insulin resistance; thus, it is imperative to characterize the chemotransduction mechanisms of this organ in order to modulate its activity and improve patient outcomes. For several years, and although controversial, cyclic adenosine monophosphate (cAMP) was considered an important player in initiating the activation of the CB. However, its relevance was partially displaced in the 90s by the emerging role of the mitochondria and molecules such as AMP-activated protein kinase and O2-sensitive K(+) channels. Neurotransmitters/neuromodulators binding to metabotropic receptors are essential to chemotransmission in the CB, and cAMP is central to this process. cAMP also contributes to raise intracellular Ca(2+) levels, and is intimately related to the cellular energetic status (AMP/ATP ratio). Furthermore, cAMP signaling is a target of multiple current pharmacological agents used in clinical practice. This review (1) provides an outline on the classical view of the cAMP-signaling pathway in the CB that originally supported its role in the O2/CO2 sensing mechanism, (2) presents recent evidence on CB cAMP neuromodulation and (3) discusses how CB activity is affected by current clinical therapies that modify cAMP-signaling, namely dopaminergic drugs, caffeine (modulation of A2A/A2B receptors) and roflumilast (PDE4 inhibitors). cAMP is key to any process that involves metabotropic receptors and the intracellular pathways involved in CB disease states are likely to involve this classical second messenger. Research examining the potential modification of cAMP levels and/or interactions with molecules associated with CB hyperactivity is currently in its beginning and this review will open doors for future explorations.

Soluble adenylyl cyclase in the locus coeruleus.

Although it has been demonstrated that the CO2-sensitivity in the locus coeruleus (LC) is mediated by changes in pH, the involvement of HCO3(-) in the CO2-detection mechanism in these neurons cannot be excluded. In the present work, we characterized sAC for the first time in the LC and we asked whether this enzyme is important in the detection of changes in HCO3(-)/CO2 levels in these neurons, using an approach that allowed us to isolate CO2 from pH stimulus. sAC mRNA expression and activity were upregulated from 0mM HCO3(-)/0% CO2 to 24 mM HCO3(-)/5% CO2 in the LC but not in the cortex of the brain. Comparing the effects of sAC and tmAC inhibitors in the LC, we observed that both tmAC and sAC contribute to the generation of cAMP during normocapnic conditions but only sAC contributed to the generation of cAMP during isohydric hypercapnia. Furthermore, activation of tmAC induced an increase in sAC expression in LC, but not cortex. sAC may be involved in CO2 sensitivity in the LC, up to its threshold of saturation, with a particular contribution of this enzyme in situations when low HCO3(-) concentrations occur. Its role should be further explored in pathological states to determine whether sAC activation with HCO3(-) alters ventilation.

Bicarbonate-sensitive soluble and transmembrane adenylyl cyclases in peripheral chemoreceptors.

Stimulation of the carotid body (CB) chemoreceptors by hypercapnia triggers a reflex ventilatory response via a cascade of cellular events, which includes generation of cAMP. However, it is not known if molecular CO2/HCO3(-) and/or H(+) mediate this effect and how these molecules contribute to cAMP production. We previously reported that the CB highly expresses HCO3(-)-sensitive soluble adenylyl cyclase (sAC). In the present study we systematically characterize the role of sAC in the CB, comparing the effect of isohydric hypercapnia (IH) in cAMP generation through activation of sAC or transmembrane-adenylyl cyclase (tmAC). Pharmacological deactivation of sAC and tmAC decreased the CB cAMP content in normocapnia and IH with no differences between these two conditions. Changes from normocapnia to IH did not effect the degree of PKA activation and the carotid sinus nerve discharge frequency. sAC and tmAC are functional in CB but intracellular elevations in CO2/HCO3(-) in IH conditions on their own are insufficient to further activate these enzymes, suggesting that the hypercapnic response is dependent on secondary acidosis.

Effect of oxygen on phosphodiesterases (PDE) 3 and 4 isoforms and PKA activity in the superior cervical ganglia.

UNLABELLED: The cAMP-protein kinase A (PKA) signaling pathway is involved in regulating the release of transmitters from neurons and other cells. Multiple phosphodiesterase (PDE) isoforms regulate this pathway, however, the pattern of isoform expression and stimulus response across tissues has not been fully characterized.Using fluorescent resonance energy transfer (FRET)-based imaging in primary superior cervical ganglia (SCG) neurons and real-time qPCR, we explored the role of PDE3 and PDE4 isoforms and oxygen tension in the activation of PKA and changes in gene expression. These primary neurons were infected with an adenovirus containing A-Kinase activity reporter (AKAR3) and assayed for responses to PDE inhibitors: rolipram (ROL, 1 µM), milrinone (MIL, 10 µM) and IBMX (100 µM), and adenylyl cyclase activator forskolin (FSK, 50 µM). Different PDE activity patterns were observed in different cells: high PDE4 activity (n = 3), high PDE3 activity (n = 3) and presence of activity of other PDEs (n = 3). Addition of PKA inhibitor H89 (10 µM) completely reversed the response. We further studied the effect of oxygen in the PKA activity induced by PDE inhibition. Both normoxia (20%O(2)/5%CO(2)) and hypoxia (0%O(2)/5%CO(2)) induced a similar increase in the FRET emission ratio (14.5 ± 0.8 and 14.7 ± 0.8, respectively).PDE3a, PDE4b and PDE4d isoforms mRNAs were highly expressed in the whole SCG with no modulation by hypoxia. CONCLUSION: Using a FRET-based PKA activity sensor, we show that primary SCG neurons can be used as a model system to dissect the contribution of different PDE isoforms in regulating cAMP/PKA signaling. The differential patterns of PDE regulation potentially represent subpopulations of ganglion cells with different physiological functions.

Effect of development on [Ca2+]i transients to ATP in petrosal ganglion neurons: a pharmacological approach using optical recording.

ATP, acting through P2X(2)/P2X(3) receptor-channel complexes, plays an important role in carotid body chemoexcitation in response to natural stimuli in the rat. Since the channels are permeable to calcium, P2X activation by ATP should induce changes in intracellular calcium ([Ca(2+)](i)). Here, we describe a novel ex vivo approach using fluorescence [Ca(2+)](i) imaging that allows screening of retrogradely labeled chemoafferent neurons in the petrosal ganglion of the rat. ATP-induced [Ca(2+)](i) responses were characterized at postnatal days (P) 5-8 and P19-25. While all labeled cells showed a brisk increase in [Ca(2+)](i) in response to depolarization by high KCl (60 mM), only a subpopulation exhibited [Ca(2+)](i) responses to ATP. ATP (250-1,000 µM) elicited one of three temporal response patterns: fast (R1), slow (R2), and intermediate (R3). At P5-8, R2 predominated and its magnitude was attenuated 44% by the P2X(1) antagonist, NF449 (10 µM), and 95% by the P2X(1)/P2X(3)/P2X(2/3) antagonist, TNP-ATP (10 µM). At P19-25, R1 and R3 predominated and their magnitudes were attenuated 15% by NF449, 66% by TNP-ATP, and 100% by suramin (100 µM), a nonspecific P2 purinergic receptor antagonist. P2X(1) and P2X(2) protein levels in the petrosal ganglion decreased with development, while P2X(3) protein levels did not change significantly. We conclude that the profile of ATP-induced P2X-mediated [Ca(2+)](i) responses changes in the postnatal period, corresponding with changes in receptor isoform expression. We speculate that these changes may participate in the postnatal maturation of chemosensitivity.

Necrostatin decreases oxidative damage, inflammation, and injury after neonatal HI.

Necrostatin-1 inhibits receptor-interacting protein (RIP)-1 kinase and programmed necrosis and is neuroprotective in adult rodent models. Owing to the prominence of necrosis and continuum cell death in neonatal hypoxia-ischemia (HI), we tested whether necrostatin was neuroprotective in the developing brain. Postnatal day (P)7 mice were exposed to HI and injected intracerebroventricularly with 0.1 µL of 80 µmol necrostatin, Nec-1, 5-(1H-Indol-3-ylmethyl)-(2-thio-3-methyl) hydantoin, or vehicle. Necrostatin significantly decreased injury in the forebrain and thalamus at P11 and P28. There was specific neuroprotection in necrostatin-treated males. Necrostatin treatment decreased necrotic cell death and increased apoptotic cell death. Hypoxia-ischemia enforced RIP1-RIP3 complex formation and inhibited RIP3-FADD (Fas-associated protein with death domain) interaction, and these effects were blocked by necrostatin. Necrostatin also decreased HI-induced oxidative damage to proteins and attenuated markers of inflammation coincidental with decreased nuclear factor-κB and caspase 1 activation, and FLIP ((Fas-associated death-domain-like IL-1ß-converting enzyme)-inhibitory protein) gene and protein expression. In this model of severe neonatal brain injury, we find that cellular necrosis can be managed therapeutically by a single dose of necrostatin, administered after HI, possibly by interrupting RIP1-RIP3-driven oxidative injury and inflammation. The effects of necrostatin treatment after HI reflect the importance of necrosis in the delayed phases of neonatal brain injury and represent a new direction for therapy of neonatal HI.

Correlation between serum caffeine levels and changes in cytokine profile in a cohort of preterm infants.

OBJECTIVE: To determine changes in cytokine levels associated with caffeine treatment in a cohort of preterm infants. STUDY DESIGN: For this observational prospective study, we collected clinical data from 26 preterm infants (≤ 30 weeks gestational age). In addition to caffeine levels, cytokine profiles in peripheral blood (PB) and tracheal aspirates (TA) were determined with enzyme-linked immunosorbent assay at birth, before and after (at 24 hours and 1 week) initiation of caffeine. Non-parametric statistics were applied. RESULTS: Included infants were 26.9 ± 1.7 weeks gestational age and weighed 985 ± 202 g. At birth, all cytokine concentrations were significantly greater in TA than PB. Serum caffeine levels were 11.1 µg/mL (interquartile range, 1.85) at approximately 24 hours post-load and 16.4 (8.7) µg/mL at 1 week on treatment. At approximately 24 hours post-load, interleukin (IL)-10 levels decreased by 47.5% (P = .01) in PB and 38.5% (P = .03) in TA, whereas other cytokine levels remained unchanged. At 1 week, caffeine levels were correlated (U-shaped) with changes in proinflammatory tumor necrosis factor-α (R(2) = 0.65; P = .0008), interleukin (IL)-1ß (R(2) = 0.73; P = .0007), and IL-6 (R(2) = 0.59; P = .003), whereas inversely correlated (linear) with the anti-inflammatory IL-10 (R(2) = 0.64; P = .0008). Altogether, caffeine, at serum levels ≥ 20 µg/mL, was associated with a proinflammatory profile after 1 week of treatment. CONCLUSIONS: Caffeine treatment for apnea of prematurity correlates with changes in cytokine profile. Caffeine levels ≥ 20 µg/mL are associated with a proinflammatory profile in our cohort of preterm infants.