Ampakines Stimulate Diaphragm Activity after Spinal Cord Injury.

Respiratory compromise after cervical spinal cord injury (SCI) is a leading cause of mortality and morbidity. Most SCIs are incomplete, and spinal respiratory motoneurons as well as proprio- and bulbospinal synaptic pathways provide a neurological substrate to enhance respiratory output. Ampakines are allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are prevalent on respiratory neurons. We hypothesized that low dose ampakine treatment could safely and effectively increase diaphragm electromyography (EMG) activity that has been impaired as a result of acute- or sub-acute cervical SCI. Diaphragm EMG was recorded using chronic indwelling electrodes in unanesthetized, freely moving rats. A spinal hemi-lesion was induced at C2 (C2Hx), and rats were studied at 4 and 14 days post-injury during room air breathing and acute respiratory challenge accomplished by inspiring a 10% O2, 7% CO2 gas mixture. Once a stable baseline recording was established, one of two different ampakines (CX717 or CX1739, 5 mg/kg, intravenous) or a vehicle (2-hydroxypropyl-beta-cyclodextrin [HPCD]) was delivered. At 4 days post-injury, both ampakines increased diaphragm EMG output ipsilateral to C2Hx during both baseline breathing and acute respiratory challenge. Only CX1739 treatment also led to a sustained (15 min) increase in ipsilateral EMG output. At 14 days post-injury, both ampakines produced sustained increases in ipsilateral diaphragm EMG output and enabled increased output during the respiratory challenge. We conclude that low dose ampakine treatment can increase diaphragm EMG activity after cervical SCI, and therefore may provide a pharmacological strategy that could be useful in the context of respiratory rehabilitation.

Countering Opioid-induced Respiratory Depression in Male Rats with Nicotinic Acetylcholine Receptor Partial Agonists Varenicline and ABT 594.

BACKGROUND: Opioids can induce significant respiratory depression when administered as analgesics for the treatment of acute, postoperative, and chronic pain. There are currently no pharmacologic means of reversing opioid-induced respiratory depression without interfering with analgesia. Further, there is a growing epidemic of opioid overdose that could benefit from therapeutic advancements. The aim of this study was to test the ability of two partial agonists of α4ß2 nicotinic acetylcholine receptors, varenicline (used clinically for smoking cessation) and ABT 594 (tebanicline, developed as an analgesic), to reduce respiratory depression induced by fentanyl, remifentanil, morphine, and a combination of fentanyl and diazepam. METHODS: Whole body plethysmographic recordings, nociception testing, and righting reflex testing were used to examine ventilation, analgesia, and sedation in adult male Sprague-Dawley rats. RESULTS: Pre-, co-, or postadministration of varenicline or ABT 594 did not alter baseline breathing but markedly reduced opioid-induced respiratory depression. Varenicline had no effect on fentanyl-induced analgesia and ABT 594 potentiated fentanyl-induced analgesia. Specifically, 10-min administration of fentanyl induced a decrease in respiratory rate to 43 ± 32% of control in vehicle group, which was alleviated by preadministration of varenicline (85 ± 14% of control, n = 8, P < 0.001) or ABT 594 (81 ± 36% of control, n = 8, P = 0.001). ABT 594 or varenicline with a low dose of naloxone (1 µg/kg), but not varenicline alone, partially reversed fentanyl-induced lethal apnea, but neither compound provided the very rapid and complete reversal of apnea achieved with high doses of naloxone (0.03 to 1 mg/kg). Administration of varenicline (n = 4, P = 0.034) or ABT 594 (n = 4, P = 0.034) prevented lethal apneas induced by the combination of fentanyl and diazepam. CONCLUSIONS: Activation of α4ß2 nicotinic acetylcholine receptors by varenicline and ABT 594 counters opioid-induced respiratory depression without interfering with analgesia.

Late Rescue Therapy with Cord-Derived Mesenchymal Stromal Cells for Established Lung Injury in Experimental Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD), the main complication of extreme prematurity, has lifelong consequences for lung health. Mesenchymal stromal cells (MSCs) prevent lung injury in experimental BPD in newborn rodents when given in the immediate neonatal period. Whether MSC therapy can restore normal lung growth after established lung injury in adulthood is clinically relevant, but currently unknown. Experimental BPD was achieved by exposing newborn rats to 95% O2 from postnatal days 4-14. Human umbilical cord-derived MSCs were intratracheally administered to rats (1 × 106cells/kg body weight) as a single dose at 3 or 6 months of age followed by assessment at 5 or 8 months of age, respectively. Lung alveolar structure and vessel density were histologically analyzed. O2-exposed rats exhibited persistent lung injury characterized by arrested alveolar growth with airspace enlargement and a lower vessel density at both 5 and 8 months of age compared with controls. Single-dose MSC treatment at 3 months partially attenuated O2-induced alveolar injury and restored vessel density at 5 months. Treatment with a single dose at 6 months did not attenuate alveolar injury or vessel density at 8 months. However, treatment with multiple MSC doses at 6, 6.5, 7, and 7.5 months significantly attenuated alveolar injury and improved vessel density at 8 months of age. Treatment of the adult BPD lung with MSCs has the potential to improve lung injury if administered in multiple doses or at an early stage of adulthood.

Regulation of breathing pattern by IL-10.

Proinflammatory cytokines like interleukin-1ß (IL-1ß) affect the control of breathing. Our aim is to determine the effect of the anti-inflammatory cytokine IL-10 οn the control of breathing. IL-10 knockout mice (IL-10-/-, n = 10) and wild-type mice (IL-10+/+, n = 10) were exposed to the following test gases: hyperoxic hypercapnia 7% CO2-93% O2, normoxic hypercapnia 7% CO2-21% O2, hypoxic hypercapnia 7% CO2-10% O2, and hypoxic normocapnia 3% CO2-10% O2. The ventilatory function was assessed using whole body plethysmography. Recombinant mouse IL-10 (rIL-10; 10 µg/kg) was administered intraperitoneally to wild-type mice (n = 10) 30 min before the onset of gas challenge. IL-10 was administered in neonatal medullary slices (10-30 ng/ml, n = 8). We found that IL-10-/- mice exhibited consistently increased frequency and reduced tidal volume compared with IL-10+/+ mice during room air breathing and in all test gases (by 23.62 to 33.2%, P < 0.05 and -36.23 to -41.69%, P < 0.05, respectively). In all inspired gases, the minute ventilation of IL-10-/- mice was lower than IL-10+/+ (by -15.67 to -22.74%, P < 0.05). The rapid shallow breathing index was higher in IL-10-/- mice compared with IL-10+/+ mice in all inspired gases (by 50.25 to 57.5%, P < 0.05). The intraperitoneal injection of rIL-10 caused reduction of the respiratory rate and augmentation of the tidal volume in room air and also in all inspired gases (by -12.22 to -29.53 and 32.18 to 45.11%, P < 0.05, respectively). IL-10 administration in neonatal rat (n = 8) in vitro rhythmically active medullary slice preparations did not affect either rhythmicity or peak amplitude of hypoglossal nerve discharge. In conclusion, IL-10 may induce a slower and deeper pattern of breathing.

Activating α4ß2 Nicotinic Acetylcholine Receptors Alleviates Fentanyl-induced Respiratory Depression in Rats.

BACKGROUND: Opioid analgesics are widely used for treatment of acute, postoperative, and chronic pain. However, activation of opioid receptors can result in severe respiratory depression. There is an unmet clinical need to develop a pharmacologic therapy to counter opioid-induced respiratory depression without interfering with analgesia. Further, additional advances to confront accidental lethal overdose with the use of fentanyl and other opioids are needed. Here, the authors test the hypothesis that activation of nicotinic receptors expressed within respiratory rhythm-generating networks would counter opioid-induced respiratory depression without compromising analgesia. METHODS: Respiratory neural discharge was measured using in vitro brainstem-spinal cord and medullary slice rat preparations. In vivo, plethysmographic recording, nociception testing, and righting reflexes were used to examine respiratory ventilation, analgesia, and sedation, respectively. RESULTS: The administration of nicotine, selective α4ß2 nicotinic receptor agonist A85380, but not α7 nicotinic receptor agonist PNU282987, reversed opioid-induced respiratory depression in neonatal pups in vitro and in vivo. In adult rats in vivo, administration of A85380 (0.03 mg/kg), but not PNU282987, provides a rapid and robust reversal of fentanyl-induced decrease in respiratory rate (93.4 ± 33.7% of control 3 min after A85380 vs. 31 ± 20.5% of control after vehicle, n = 8 each, P < 0.001), without marked side effects. The coadministration of A85380 (0.06 mg/kg) with fentanyl or remifentanil markedly reduced respiratory depression and apneas, and enhanced the fentanyl-induced analgesia, as evidenced by increased paw withdrawal latency in Hargreaves plantar test (14.4 ± 2.8 s vs. vehicle: 11.3 ± 2.4 s, n = 8 each, P = 0.013) and decreased formalin-induced nocifensive duration (2.5 ± 2.4 min vs. vehicle: 5.4 ± 2.7 min, n = 8 each, P = 0.029). CONCLUSIONS: The novel strategy of targeting α4ß2 nicotinic acetylcholine receptors has the potential for advancing pain control and reducing opioid-induced respiratory depression and overdose.

Cardiorespiratory pathogenesis of sickle cell disease in a mouse model.

The nature and development of cardiorespiratory impairments associated with sickle cell disease are poorly understood. Given that the mechanisms of these impairments cannot be addressed adequately in clinical studies, we characterized cardiorespiratory pathophysiology from birth to maturity in the sickle cell disease SAD mouse model. We identified two critical phases of respiratory dysfunction in SAD mice; the first prior to weaning and the second in adulthood. At postnatal day 3, 43% of SAD mice showed marked apneas, anemia, and pulmonary vascular congestion typical of acute chest syndrome; none of these mice survived to maturity. The remaining SAD mice had mild lung histological changes in room air with an altered respiratory pattern, seizures, and a high rate of death in response to hypoxia. Approximately half the SAD mice that survived to adulthood had an identifiable respiratory phenotype including baseline tachypnea at 7-8 months of age, restrictive lung disease, pulmonary hypertension, cardiac enlargement, lower total lung capacity, and pulmonary vascular congestion. All adult SAD mice demonstrated impairments in exercise capacity and response to hypoxia, with a more severe phenotype in the tachypneic mice. The model revealed distinguishable subgroups of SAD mice with cardiorespiratory pathophysiology mimicking the complications of human sickle cell disease.

Mechanistic Studies of Capsaicin-Induced Apnea in Rodents.

Inhalation of capsaicin-based sprays can cause central respiratory depression and lethal apneas. There are contradictory reports regarding the sites of capsaicin action. Furthermore, an understanding of the neurochemical mechanisms underlying capsaicin-induced apneas and the development of pharmacological interventions is lacking. The main objectives of this study were to perform a systematic study of the mechanisms of action of capsaicin-induced apneas and to provide insights relevant to pharmacological intervention. In vitro and in vivo rat and transient receptor potential vanilloid superfamily member 1 (TRPV1)-null mouse models were used to measure respiratory parameters and seizure-like activity in the presence of capsaicin and compounds that modulate glutamatergic neurotransmission. Administration of capsaicin to in vitro and in vivo rat and wild-type mouse models induced dose-dependent apneas and the production of seizure-like activity. No significant changes were observed in TRPV1-null mice or rat medullary slice preparations. The capsaicin-induced effects were inhibited by the TRPV1 antagonist capsazepine, amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonists CNQX, NBQX, perampanel, and riluzole, a drug that inhibits glutamate release and increases glutamate uptake. The capsaicin-induced effects on breathing and seizure-like activity were accentuated by positive allosteric modulators of the AMPA receptors, CX717 and cyclothiazide. To summarize, capsaicin-induced apneas and seizure-like behaviors are mediated via TRPV1 activation acting at lung afferents, spinal cord-ascending tracts, and medullary structures (including nucleus tractus solitarius). AMPA receptor-mediated conductances play an important role in capsaicin-induced apneas and seizure-like activity. A pharmaceutical strategy targeted at reducing AMPA receptor-mediated glutamatergic signaling may reduce capsaicin-induced deleterious effects.

Developmental plasticity of phrenic motoneuron and diaphragm properties with the inception of inspiratory drive transmission in utero.

The review outlines data consistent with the hypothesis that inspiratory drive transmission that generates fetal breathing movements (FBMs) is essential for the developmental plasticity of phrenic motoneurons (PMNs) and diaphragm musculature prior to birth. A systematic examination during the perinatal period demonstrated a very marked transformation of PMN and diaphragm properties coinciding with the onset and strengthening of inspiratory drive and FBMs in utero. This included studies of age-dependent changes of: i) morphology, neuronal coupling, passive and electrophysiological properties of PMNs; ii) rhythmic inspiratory activity in vitro; iii) FBMs generated in vivo detected by ultrasonography; iv) contractile and end-plate potential properties of diaphragm musculature. We also propose how the hypothesis can be further evaluated with studies of perinatal hypoglossal motoneuron-tongue musculature and the use of Dbx1 null mice that provide an experimental model lacking descending inspiratory drive transmission in utero.

Ampakine CX1942 attenuates opioid-induced respiratory depression and corrects the hypoxaemic effects of etorphine in immobilized goats (Capra hircus).

OBJECTIVES: To determine whether CX1942 reverses respiratory depression in etorphine-immobilized goats, and to compare its effects with those of doxapram hydrochloride. STUDY DESIGN: A prospective, crossover experimental trial conducted at 1753 m.a.s.l. ANIMALS: Eight adult female Boer goats (Capra hircus) with a mean ± standard deviation mass of 27.1 ± 1.6 kg. METHODS: Following immobilization with 0.1 mg kg(-1) etorphine, goats received one of doxapram, CX1942 or sterile water intravenously, in random order in three trials. Respiratory rate, ventilation and tidal volume were measured continuously. Arterial blood samples for the determination of PaO2 , PaCO2 , pH and SaO2 were taken 2 minutes before and then at 5 minute intervals after drug administration for 25 minutes. RESULTS: Doxapram corrected etorphine-induced respiratory depression but also led to arousal and hyperventilation at 2 minutes after its administration, as indicated by the low PaCO2 (27.8 ± 4.5 mmHg) and ventilation of 5.32 ± 5.24 L minute(-1) above pre-immobilization values. CX1942 improved respiratory parameters and corrected etorphine's hypoxaemic effects more gradually than did doxapram, with a more sustained improvement in PaO2 and SaO2 in comparison with the control trial. CONCLUSIONS: CX1942 attenuated opioid-induced respiratory depression and corrected the hypoxaemic effects of etorphine in immobilized goats. CLINICAL RELEVANCE: Ampakines potentially offer advantages over doxapram, a conventional treatment, in reversing etorphine-induced respiratory depression without causing unwanted side effects, particularly arousal, in immobilized animals.

Muscle dysfunction caused by loss of Magel2 in a mouse model of Prader-Willi and Schaaf-Yang syndromes.

Prader-Willi syndrome is characterized by severe hypotonia in infancy, with decreased lean mass and increased fat mass in childhood followed by severe hyperphagia and consequent obesity. Scoliosis and other orthopaedic manifestations of hypotonia are common in children with Prader-Willi syndrome and cause significant morbidity. The relationships among hypotonia, reduced muscle mass and scoliosis have been difficult to establish. Inactivating mutations in one Prader-Willi syndrome candidate gene, MAGEL2, cause a Prader-Willi-like syndrome called Schaaf-Yang syndrome, highlighting the importance of loss of MAGEL2 in Prader-Willi syndrome phenotypes. Gene-targeted mice lacking Magel2 have excess fat and decreased muscle, recapitulating altered body composition in Prader-Willi syndrome. We now demonstrate that Magel2 is expressed in the developing musculoskeletal system, and that loss of Magel2 causes muscle-related phenotypes in mice consistent with atrophy caused by altered autophagy. Magel2-null mice serve as a preclinical model for therapies targeting muscle structure and function in children lacking MAGEL2 diagnosed with Prader-Willi or Schaaf-Yang syndrome.

Novel Therapies for the Treatment of Central Sleep Apnea.

Neurophysiologically, central apnea is due to a temporary cessation of respiratory rhythmogenesis in medullary respiratory networks. Central apneas occur in several disorders and result in pathophysiological consequences, including arousals and desaturation. The 2 most common causes in adults are congestive heart failure and chronic use of opioids to treat pain. Under such circumstances, diagnosis and treatment of central sleep apnea may improve quality of life, morbidity, and mortality. This article discusses recent developments in the treatment of central sleep apnea in heart failure and opioids use.

G-protein-gated Inwardly Rectifying Potassium Channels Modulate Respiratory Depression by Opioids.

BACKGROUND: Drugs acting on µ-opioid receptors (MORs) are widely used as analgesics but present side effects including life-threatening respiratory depression. MORs are G-protein-coupled receptors inhibiting neuronal activity through calcium channels, adenylyl cyclase, and/or G-protein-gated inwardly rectifying potassium (GIRK) channels. The pathways underlying MOR-dependent inhibition of rhythmic breathing are unknown. METHODS: By using a combination of genetic, pharmacological, and physiological tools in rodents in vivo, the authors aimed to identify the role of GIRK channels in MOR-mediated inhibition of respiratory circuits. RESULTS: GIRK channels were expressed in the ventrolateral medulla, a neuronal population regulating rhythmic breathing, and GIRK channel activation with flupirtine reduced respiratory rate in rats (percentage of baseline rate in mean ± SD: 79.4 ± 7.4%, n = 7), wild-type mice (82.6 ± 3.8%, n = 3), but not in mice lacking the GIRK2 subunit, an integral subunit of neuronal GIRK channels (GIRK2, 101.0 ± 1.9%, n = 3). Application of the MOR agonist [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO) to the ventrolateral medulla depressed respiratory rate, an effect partially reversed by the GIRK channel blocker Tertiapin-Q (baseline: 42.1 ± 7.4 breath/min, DAMGO: 26.1 ± 13.4 breath/min, Tertiapin-Q + DAMGO: 33.9 ± 9.8 breath/min, n = 4). Importantly, DAMGO applied to the ventrolateral medulla failed to reduce rhythmic breathing in GIRK2 mice (percentage of baseline rate: 103.2 ± 12.1%, n = 4), whereas it considerably reduced rate in wild-type mice (62.5 ± 17.7% of baseline, n = 4). Respiratory rate depression by systemic injection of the opioid analgesic fentanyl was markedly reduced in GIRK2 (percentage of baseline: 12.8 ± 15.8%, n = 5) compared with wild-type mice (72.9 ± 27.3%). CONCLUSIONS: Overall, these results identify that GIRK channels contribute to respiratory inhibition by MOR, an essential step toward understanding respiratory depression by opioids.

Stimulation of Respiratory Motor Output and Ventilation in a Murine Model of Pompe Disease by Ampakines.

Pompe disease results from a mutation in the acid α-glucosidase gene leading to lysosomal glycogen accumulation. Respiratory insufficiency is common, and the current U.S. Food and Drug Administration-approved treatment, enzyme replacement, has limited effectiveness. Ampakines are drugs that enhance α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor responses and can increase respiratory motor drive. Recent work indicates that respiratory motor drive can be blunted in Pompe disease, and thus pharmacologic stimulation of breathing may be beneficial. Using a murine Pompe model with the most severe clinical genotype (the Gaa(-/-) mouse), our primary objective was to test the hypothesis that ampakines can stimulate respiratory motor output and increase ventilation. Our second objective was to confirm that neuropathology was present in Pompe mouse medullary respiratory control neurons. The impact of ampakine CX717 on breathing was determined via phrenic and hypoglossal nerve recordings in anesthetized mice and whole-body plethysmography in unanesthetized mice. The medulla was examined using standard histological methods coupled with immunochemical markers of respiratory control neurons. Ampakine CX717 robustly increased phrenic and hypoglossal inspiratory bursting and reduced respiratory cycle variability in anesthetized Pompe mice, and it increased inspiratory tidal volume in unanesthetized Pompe mice. CX717 did not significantly alter these variables in wild-type mice. Medullary respiratory neurons showed extensive histopathology in Pompe mice. Ampakines stimulate respiratory neuromotor output and ventilation in Pompe mice, and therefore they have potential as an adjunctive therapy in Pompe disease.

Ampakines enhance weak endogenous respiratory drive and alleviate apnea in perinatal rats.

RATIONALE: Apnea of prematurity, which is prevalent among infants born at less than 34 weeks gestation, is treated with caffeine, theophylline, or aminophylline. However, not all newborns respond adequately to, or tolerate, methylxanthine administration, and thus alternative pharmacological therapies are required. OBJECTIVES: Rodent models are used to test the hypothesis that the ampakine CX1739, a positive allosteric modulator of amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors, strengthens perinatal respiratory drive and reduces apneas. We also provide a systematic study of the effects of caffeine for comparison. METHODS: Respiratory neural activity was recorded from brainstem-spinal cord in vitro perinatal rat preparations, and [Formula: see text]e was recorded in newborn rat pups using whole-body plethysmography under normoxic and hypoxic conditions. MEASUREMENTS AND MAIN RESULTS: Using in vitro brainstem-spinal cord preparations, we found that CX1739 (10-100 µM) dose-dependently increases the frequency of respiratory activity generated by fetal and newborn rat preparations under normoxic and hypoxic conditions. Plethysmographic recordings in vivo from Postnatal Day 0 rats demonstrated that CX1739 (10 mg/kg) increases the frequency and regularity of ventilation, reduces apneas, and protects against hypoxia-induced respiratory depression. CONCLUSIONS: The net effect of ampakine enhancement of respiratory drive in perinatal rodents is a marked increase in ventilation and the regularity of respiratory patterns in perinatal rat preparations. Importantly, from the perspective of clinical applications, CX1739 readily crosses the blood-brain barrier, is metabolically stable, and has passed through phase I and II clinical trials in adults.

5-HT1A receptor agonist Befiradol reduces fentanyl-induced respiratory depression, analgesia, and sedation in rats.

BACKGROUND: There is an unmet clinical need to develop a pharmacological therapy to counter opioid-induced respiratory depression without interfering with analgesia or behavior. Several studies have demonstrated that 5-HT1A receptor agonists alleviate opioid-induced respiratory depression in rodent models. However, there are conflicting reports regarding their effects on analgesia due in part to varied agonist receptor selectivity and presence of anesthesia. Therefore the authors performed a study in rats with befiradol (F13640 and NLX-112), a highly selective 5-HT1A receptor agonist without anesthesia. METHODS: Respiratory neural discharge was measured using in vitro preparations. Plethysmographic recording, nociception testing, and righting reflex were used to examine respiratory ventilation, analgesia, and sedation, respectively. RESULTS: Befiradol (0.2 mg/kg, n = 6) reduced fentanyl-induced respiratory depression (53.7 ± 5.7% of control minute ventilation 4 min after befiradol vs. saline 18.7 ± 2.2% of control, n = 9; P < 0.001), duration of analgesia (90.4 ± 11.6 min vs. saline 130.5 ± 7.8 min; P = 0.011), duration of sedation (39.8 ± 4 min vs. saline 58 ± 4.4 min; P = 0.013); and induced baseline hyperventilation, hyperalgesia, and "behavioral syndrome" in nonsedated rats. Further, the befiradol-induced alleviation of opioid-induced respiratory depression involves sites or mechanisms not functioning in vitro brainstem-spinal cord and medullary slice preparations. CONCLUSIONS: The reversal of opioid-induced respiratory depression and sedation by befiradol in adult rats was robust, whereas involved mechanisms are unclear. However, there were adverse concomitant decreases in fentanyl-induced analgesia and altered baseline ventilation, nociception, and behavior.

Glial TLR4 signaling does not contribute to opioid-induced depression of respiration.

Opioids activate glia in the central nervous system in part by activating the toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) complex. TLR4/MD2-mediated activation of glia by opioids compromises their analgesic actions. Glial activation is also hypothesized as pivotal in opioid-mediated reward and tolerance and as a contributor to opioid-mediated respiratory depression. We tested the contribution of TLR4 to opioid-induced respiratory depression using rhythmically active medullary slices that contain the pre-Bötzinger Complex (preBötC, an important site of respiratory rhythm generation) and adult rats in vivo. Injection with DAMGO (µ-opioid receptor agonist; 50 µM) or bath application of DAMGO (500 nM) or fentanyl (1 µM) slowed frequency recorded from XII nerves to 40%, 40%, or 50% of control, respectively. This DAMGO-mediated frequency inhibition was unaffected by preapplication of lipopolysaccharides from Rhodobacter sphaeroides (a TLR4 antagonist, 2,000 ng/ml) or (+)naloxone (1-10 µM, a TLR4-antagonist). Bath application of (-)naloxone (500 nM; a TLR4 and µ-opioid antagonist), however, rapidly reversed the opioid-mediated frequency decrease. We also compared the opioid-induced respiratory depression in slices in vitro in the absence and presence of bath-applied minocycline (an inhibitor of microglial activation) and in slices prepared from mice injected (ip) 18 h earlier with minocycline or saline. Minocycline had no effect on respiratory depression in vitro. Finally, the respiratory depression evoked in anesthetized rats by tail vein infusion of fentanyl was unaffected by subsequent injection of (+)naloxone, but completely reversed by (-)naloxone. These data indicate that neither activation of microglia in preBötC nor TLR4/MD2-activation contribute to opioid-induced respiratory depression.

Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development.

Current concepts on the pathogenesis and etiology of congenital diaphragmatic hernia.

This review outlines research that has advanced our understanding of the pathogenesis and etiology of congenital diaphragmatic hernia (CDH). The majority of CDH cases involve incomplete formation of the posterolateral portion of the diaphragm, clinically referred to as a Bochdalek hernia. The hole in the diaphragm allows the abdominal viscera to invade the thoracic cavity, thereby impeding normal lung development. As a result, newborns with CDH suffer from a combination of severe pulmonary hypoplasia and pulmonary hypertension. Despite advances in neonatal intensive care, mortality and serious morbidity remain high. Systematic studies using rat and transgenic mouse models in conjunction with analyses of human tissue are providing insights into the embryological origins of the diaphragmatic defect associated with CDH and abnormalities of developmentally regulated signaling cascades.

Coadministration of the AMPAKINE CX717 with propofol reduces respiratory depression and fatal apneas.

BACKGROUND: Propofol (2,6-diisopropylphenol) is used for the induction and maintenance of anesthesia in human and veterinary medicine. Propofol's disadvantages include the induction of respiratory depression and apnea. Here, the authors report a clinically feasible pharmacological solution for reducing propofol-induced respiratory depression via a mechanism that does not interfere with anesthesia. Specifically, they test the hypothesis that the AMPAKINE CX717, which has been proven metabolically stable and safe for human use, can prevent and rescue from propofol-induced severe apnea. METHODS: The actions of propofol and the AMPAKINE CX717 were measured via (1) ventral root recordings from newborn rat brainstem-spinal cord preparations, (2) phrenic nerve recordings from an adult mouse in situ working heart-brainstem preparation, and (3) plethysmographic recordings from unrestrained newborn and adult rats. RESULTS: In vitro, respiratory depression caused by propofol (2 µM, n = 11, mean ± SEM, 41 ± 5% of control frequency, 63 ± 5% of control duration) was alleviated by CX717 (n = 4, 50-150 µM). In situ, a decrease in respiratory frequency (44 ± 9% of control), phrenic burst duration (66 ± 7% of control), and amplitude (78 ± 5% of control) caused by propofol (2 µM, n = 5) was alleviated by coadministration of CX717 (50 µM, n = 5). In vivo, pre- or coadministration of CX717 (20-25mg/kg) with propofol markedly reduced propofol-induced respiratory depression (n = 7; 20mg/kg) and propofol-induced lethal apnea (n = 6; 30 mg/kg). CONCLUSIONS: Administration of CX717 before or in conjunction with propofol provides an increased safety margin against profound apnea and death.

The rhythmic, transverse medullary slice preparation in respiratory neurobiology: contributions and caveats.

Our understanding of the sites and mechanisms underlying rhythmic breathing as well as the neuromodulatory control of respiratory rhythm, pattern, and respiratory motoneuron excitability during perinatal development has advanced significantly over the last 20 years. A major catalyst was the development in 1991 of the rhythmically-active medullary slice preparation, which provided precise mechanical and chemical control over the network as well as enhanced physical and optical access to key brainstem regions. Insights obtained in vitro have informed multiple mechanistic hypotheses. In vivo tests of these hypotheses, performed under conditions of reduced control and precision but more obvious physiological relevance, have clearly established the significance for respiratory neurobiology of the rhythmic slice preparation. We review the contributions of this preparation to current understanding/concepts in respiratory control, and outline the limitations of this approach in the context of studying rhythm and pattern generation, homeostatic control mechanisms and murine models of human genetic disorders that feature prominent breathing disturbances.