ABSTRACTderived from the 129/sv strain (Clontech) was screened using a mouse D3 cDNA probe (17). A positive clone encompassing exon 2 of the murine D3 gene was isolated and further characterized. A 7-kb Xho I-Asp718 fragment was engineered for targeted mutagenesis by introducing the GKNeo cassette (16) in antisense orientation at the Sal I site in exon 2 (17). Integration of sequences derived from the pGKNeo cassette generates a novel open reading frame, resulting in the following peptide sequence appended after Arg-148: PASDGIRT-WQNNTENEVYVEQRLLISFFRL Opal (Stop). The sequence of the mutant allele was confirmed by direct sequencing of reverse transcription-PCR (rPCR) products derived from brain mRNAs of -/-and +/-mice (data not shown).Transfection ofES Cells and Embryo Manipulations. J-1 ES cells (a kind gift of R. Jaenisch, Massachussetts Institute of Technology) at passage 13 were grown on mitomycin C-treated embryonic fibroblasts derived from a homozygous neomycin (Neo)-resistant transgenic mouse (16). Cells (2 x 107) were electroporated in a 1-ml cuvette (path length-0.2 cm) at 0.4 kV and 25 ,uF. Cells were plated onto 40 gelatin-coated Petri dishes (6 cm) on embryonic feeder cells. Selection with G418 (0.3 mg/ml; active concentration of 0.66 ,vg/mg of dry powder; GIBCO) was applied 24 hr after plating and was continued for 7-9 days. Individual Neo-resistant colonies were picked using a dissection microscope and expanded as described (16). Genomic DNA was prepared from an aliquot of cells for each clone using previously described techniques and analyzed by Southern blotting (18). Recovery, microinjection, and transfer of 3.5 day postcoitus embryos was performed as described (16).
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 lL of 80 lmol 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-jB and caspase 1 activation, and FLIP ((Fas-associated death-domain-like IL-1b-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.
Objective To determine if oral clonidine would reduce the duration of opioid detoxification for neonatal abstinence syndrome. Methods Infants with intrauterine exposure to methadone or heroin and neonatal abstinence syndrome (2 consecutive modified Finnegan scores of ≥9) were enrolled at 2 hospitals during 2002–2005 and followed until final hospital discharge. All enrolled infants (80) received oral diluted tincture of opium according to a standardized algorithm and were randomly assigned to receive oral clonidine (1 μg/kg every 4 hours) (40 infants) or placebo (40 infants). Primary outcome was duration of opioid therapy. Secondary outcomes included the amount of opium required to control symptoms, number of treatment failures, and differences in blood pressure, heart rate, and oxygen saturation. Results The median length of therapy was 27% shorter in the clonidine group (11 [95% confidence interval: 8–15 days]) than in the placebo group (15 days [95% confidence interval: 12–17 days]). In the clonidine group, 7 infants required restarting opium after initial discontinuation versus none in the placebo group, with the total length of treatment/observation remaining significantly less in the clonidine group. Higher dosages of opium were required by 40% of the infants in the placebo group versus 20% in the clonidine group. Treatment failures occurred in 12.5% of the infants in the placebo group versus none in the clonidine group. Hypertension, hypotension, bradycardia, or desaturations did not occur in either group. Three infants in the clonidine group died as a result of myocarditis, sudden infant death syndrome, and homicide, all after hospital discharge and before 6 months of age. Conclusions In this randomized, double-blind trial, adding clonidine to standard opioid therapy for detoxification from in utero exposure to methadone or heroin reduced the duration of pharmacotherapy for neonatal abstinence without causing short-term adverse cardiovascular outcomes. A larger trial is indicated to determine long-term safety.
D1- and D2-dopamine receptor-mediated regulation of immediate early gene levels in identified populations of neurons in the striatum was examined with quantitative in situ hybridization histochemical techniques. Levels of messenger RNA (mRNA) encoding the immediate early genes zif268 and c-fos were examined in two experiments in rats with unilateral lesions of the nigrostriatal dopamine pathway. In a dose- response study, animals were treated with doses of 0.5, 1.0, and 1.5 mg/kg of the D1 agonist SKF-38393 either alone or in combination with the D2 agonist quinpirole (1 mg/kg). Levels of immediate early gene mRNAs 60 min following drug treatments showed a dose-related increase to the D1 agonist alone and a potentiation to combined D1 and D2 against treatment. In a second experiment, in animals receiving 1 mg/kg SKF-38393 either alone or in combination with 1 mg/kg quinpirole, the level of zif268 mRNA was measured with a double-labeling method in striatal neurons containing enkephalin mRNA, a marker of D2-containing neurons, and in neurons not containing enkephalin, putative D1- containing neurons. In the dopamine-depleted striatum, D1 agonist treatment alone did not affect enkephalin-positive neurons but significantly elevated zif268 mRNA levels in nearly all enkephalin- negative neurons. Combined D1 and D2 agonist treatment further increased zif268 mRNA levels in this population of enkephalin-negative neurons and decreased zif-268 mRNA levels in enkephalin-positive neurons. These data indicate that the synergistic response to combined D1- and D2-receptor stimulation is mediated by interneuronal interactions involving the activation of D1 and D2 receptors on separate populations of striatal neurons.
The peripheral arterial chemoreceptors of the carotid body participate in the ventilatory responses to hypoxia and hypercapnia, the arousal responses to asphyxial apnea, and the acclimatization to high altitude. In response to an excitatory stimuli, glomus cells in the carotid body depolarize, their intracellular calcium levels rise, and neurotransmitters are released from them. Neurotransmitters then bind to autoreceptors on glomus cells and postsynaptic receptors on chemoafferents of the carotid sinus nerve. Binding to inhibitory or excitatory receptors on chemoafferents control the electrical activity of the carotid sinus nerve, which provides the input to respiratory-related brainstem nuclei. We and others have used gene expression in the carotid body as a tool to determine what neurotransmitters mediate the response of peripheral arterial chemoreceptors to excitatory stimuli, specifically hypoxia. Data from physiological studies support the involvement of numerous putative neurotransmitters in hypoxic chemosensitivity. This article reviews how in situ hybridization histochemistry and other cellular localization techniques confirm, refute, or expand what is known about the role of dopamine, norepinephrine, substance P, acetylcholine, adenosine, and ATP in chemotransmission. In spite of some species differences, review of the available data support that 1). dopamine and norepinephrine are synthesized and released from glomus cells in all species and play an inhibitory role in hypoxic chemosensitivity; 2). substance P and acetylcholine are not synthesized in glomus cells of most species but may be made and released from nerve fibers innervating the carotid body in essentially all species; 3). adenosine and ATP are ubiquitous molecules that most likely play an excitatory role in hypoxic chemosensitivity.
Caffeine, a nonspecific adenosine receptor (AR) antagonist is widely used to treat apnea of prematurity. Because adenosine modulates multiple biologic processes including inflammation, we hypothesized that AR blockade by caffeine would increase cytokine release from neonatal monocytes. Using cord blood monocytes (CBM), we investigated 1) the changes in AR mRNA profile by real time quantitative reverse-transcription polymerase-chain-reaction (qRT-PCR) and protein expression (western blot) after in vitro culture, caffeine or lipopolysaccharide (LPS) exposure, and 2) the modulation of cytokine release and cyclic adenosine monophosphate (cAMP) production by enzyme-linked immunosorbent assay (ELISA) induced by caffeine and specific AR antagonists: DPCPX(A 1 R), ZM241385(A 2a R), MRS1754(A 2b R), and MRS1220(A 3 R). After 48 h in culture, A 2a R and A 2b R gene expression increased 1.9 (p ϭ 0.04) and 2.5-fold (p ϭ 0.003), respectively. A 1 R protein expression directly correlated with increasing LPS concentrations (p ϭ 0.01), with minimal expression preexposure. Only caffeine (50 M) and DPCPX (10 nM) decreased tumor necrosis factor-alpha (TNF-␣) release from LPS activated-CBM by 20 and 25% (p ϭ 0.01) and TNF-␣ gene expression by 30 and 50%, respectively, in conjunction with a Ն2-fold increase in cAMP (p Ͻ 0.05). AR blockade did not modulate other measured cytokines. The induction of A 1 R after LPS exposure suggests an important role of this receptor in the control of inflammation in neonates. Our findings also suggest that caffeine, via A 1 R blockade, increases cAMP production and inhibits pretranscriptional TNF-␣ production by CBM. (Pediatr Res 65: 203-208, 2009) C affeine (1,3,11 trimethylxantine) is a stimulant widely used in neonatology to treat apnea of prematurity (1). At therapeutic range (5 to 15 g/mL), caffeine blocks A 1 and A 2a adenosine receptors (ARs) stimulating ventilation (2-4). Recently, caffeine has also been linked to a decrease in the incidence of bronchopulmonary dysplasia and cerebral palsy in extremely premature infants (5,6), although the mechanisms explaining these findings have not been elucidated.The natural ligand for ARs, adenosine, has a crucial role in multiple biologic processes including inflammation (7,8). The increase in tumor necrosis factor-alpha (TNF-␣) release by adult peripheral blood monocytes (PBM) in response to lipopolysaccharide (LPS) exposure can be abolished by pretreatment with A 2a R agonists (9,10). Adenosine binding to A 1 R (11,12) and A 3 R (13,14) also modulates TNF-␣ release from adult monocytes, whereas A 2b R appears to have little effect (10).Little is known about AR expression on neonatal monocytes and the role of caffeine in modulating cytokine release. We hypothesized that caffeine blockade of ARs on neonatal monocytes would increase the release of cytokines in response to LPS. To test this hypothesis, we used cord blood monocytes (CBM) from full-term infants to 1) characterize the changes in AR mRNA profile and protein expression after 48 h in ...
To determine how maximal inspiratory airflow (VImax) is modulated by changes in airway neuromuscular activity, we analyzed pressure-flow relationships obtained during inspiration and expiration in isolated upper airways of anesthetized hyperoxic dogs at different levels of CO2. Inspiratory airflow (VI), hypopharyngeal pressure (Php), pharyngeal pressure at the flow-limiting site (FLS), and alae nasi (AN) and genioglossus (GG) electromyographic (EMG) activity were recorded while VI limitation was produced by rapidly lowering Php until VI plateaued at VImax. VImax and its mechanical determinants, pharyngeal critical pressure (Pcrit) and nasal resistance (Rn) upstream to the FLS, were measured. During hypercapnia (high CO2), VImax increased significantly during inspiration (217.3) and expiration (184.1%). These increases were associated with significant increases in phasic but not tonic AN and GG activity. They were also associated with decreases in Pcrit from -6.2 +/- 1.6 (SE) at hypocapnia to -9.3 +/- 3.0 and -11.8 +/- 3.4 cmH2O at high CO2 during expiration and inspiration, respectively. No significant changes in Rn occurred. When phasic neuromuscular activity was abolished by complete neuromuscular blockade in three dogs, these increases in VImax and decreases in Pcrit at high CO2 were eliminated. When phasic EMG activity was accentuated in four vagotomized dogs, significant increases in VImax and decreases in Pcrit were demonstrated during inspiration vs. expiration at high CO2. These findings indicate that upper airway neuromuscular activity increases VImax in the isolated upper airway by decreasing collapsibility (Pcrit) at the FLS site when neuromuscular activity is stimulated by hypercapnia.
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